Anti cd4 antibodies to prevent in particular graft -versus - host - disease (gvhd)

ABSTRACT

The present invention relates to, among others, an in vitro method of modifying a cell graft containing immune cells comprising the steps of incubating a cell graft containing immune cells with an anti CD4 antibody wherein said incubating is carried out for from 1 minute to 7 days, b) removing unbound antibody from said graft; as well as to corresponding modified grafts and uses. The invention further relates to the modification of antibodies reactive to the CD4 human leukocyte antigen to provide anti-CD4 antibodies that have a reduced number of potential T-cell epitopes but retain the ability to bind to CD4, such as to an anti human CD4-antibody comprising a heavy chain immunoglobulin variable domain (VH) and a light chain immunoglobulin variable domain (VL), wherein at least one T cell epitope located outside the CDRs of said immunoglobulin variable domains is removed from said immunoglobulin variable domains. Preferably, the specificity and mode of action of the anti-CD4 antibodies are not affected by the modification(s).

The invention relates to the field of grafts and transplantationsthereof. In particular, the invention relates to modified grafts,methods of obtaining same, as well as related uses. Among others, theinvention relates to grafts containing immunocompetent viable cells.

In an additional aspect, the present invention relates to modified antihuman CD4-antibodies, in which the immune characteristic is modified bymeans of a reduced number of T cell epitopes, and to relatedsubject-matter.

BACKGROUND OF THE INVENTION

Today, allogeneic hematopoietic stem cell transplantation (HSCT) is theonly curative treatment for many patients with hematologicalmalignancies. Bone marrow (Aschan, 2006), peripheral mobilized stemcells (Bacigalupo et al., 2002) and umbilical cord blood (Kestendjievaet al., 2008) are the common sources for HSCT. Despite the use of highlysophisticated therapeutic approaches, HSCT is still associated with aconsiderable mortality caused by a number of complications such as graftversus host disease (GvHD), infectious diseases, veno-occlusive disease,donor graft rejection, and relapses of the underlying diseases.

The use of conventionally immunosuppressive drugs leads to a suppressionof the entire immune system, which enhances the possibility forinfections or development of malignant tumors. Also in some cases, theeffectiveness of these drugs can be reduced or even abrogated. Forexample, steroid refractory GvHD is one of the major problems followingallogeneic hematopoietic stem cell transplantation (Auletta et al.,2009; von Bonin et al., 2009). For treatment of GvHD, immunosuppressivestrategies against key elements of T-cell reactions were alreadyperformed (von Bonin et al., 2009). However, because of the high numbersof patients, these strategies were mainly used in rheumatology (Kamedaet al., 2009; Senolt et al., 2009) or for patients after kidneytransplantation. For therapy of acute GvHD, most experiences areavailable for OKT3® (Benekli M et al., 2006; Knop et al., 2007) orinterleukin 2 receptor antibodies (Chen et al., 2004; Ji et al., 2005),and for chronic GvHD with anti CD20 antibodies (Bates et al., 2009).However, these antibodies acute GvHD, most experiences are available forOKT3® (Benekli M et al., 2006; Knop et al., 2007) or interleukin 2receptor antibodies (Chen et al., 2004; Ji et al., 2005), and forchronic GvHD with anti CD20 antibodies (Bates et al., 2009). However,these antibodies can be associated with less long-term success andtoxicity because of appearance of infectious complications. The use ofmonoclonal antibodies for clinical application was restricted because ofthe missing humanization. Murine antibodies or antibodies from otherspecies are huge molecules with a molecular weight in the range of 150kDa that may be highly immunogenic in humans. After application ofmurine anti human monoclonal antibodies, life-threatening andanaphylactic complications were observed (Chester et al., 1995). Also,the immunogenic potential of the antibodies depends from their peptidestructure. IgG4 isotypes, for example, are less immunogenic than IgG1isotypes because of the low potential for complement activation.Besides, the humanization of antibodies leads to chimeric isotypes thatare less immunogenic than their originally murine counterparts (Hosonoet al., 1992). Up to date, there are no clear data that show thattotally human antibodies have clinically advantages compared to chimericantibodies.

Accordingly, the investigation of alternative or improved therapeuticapproaches or procedures including the use of new cell sources, thetreatment with antibodies or other biologicals are still in need.

One possible approach focuses on CD4 positive T helper cells. Said cellscoordinate both the pathological and the physiological immune reactionin the human body. Influencing CD4 positive T helper cells byapplication of anti CD4 antibodies should, therefore, lead to a targetedmodulation of the immune system.

Previously, the murine anti human CD4 monoclonal antibody Max16H5 (IgG1)was used in clinical application in patients with auto-immune diseasesor as a protective therapy against transplant rejection (Chatenoud etal., 1995; Emmrich et al., 1991a; Emmrich et al., 1991b). Furthermore,in human kidney transplantation, Max16H5 (IgG1) had the potential toeffectively reduce graft rejection (Reinke et al., 1991; Reinke et al.,1995). The application of anti CD4 specific monoclonal antibodies maynot only result in suppression of immune activity but also in theinduction of tolerance against tetanus toxoid in an triple transgeneicmouse model (Laub et al., 2002). The induction of tolerance by a ratmonoclonal antibody has also been demonstrated (Kohlhaw et al., 2001).Said monoclonal antibody Max16H5 is also disclosed in EP 1 454 137,which is incorporated herein by reference and which, among others,relates to the use of a labeled ligand having specificity for the humanCD4 molecule to produce an in vivo diagnostic agent. CD4+ molecules on Thelper cells bind directly to constant regions of HLA molecules onantigen presenting cells (APCs) to allow a complete T cell activation.To interfere with this binding by non depleting monoclonal antibodiesmay inhibit this activation by a total steric blockage, by shortening ofcell-cell contact between APC and T cell (Fehervari et al., 2002) or byinduction of negative signals by inhibition of protein tyrosinephosphorylation (Harding et al., 2002) or induction of T cell anergy(Madrenas et al., 1996). Here, Fehérvári at al. and Harding et al. donot disclose the methods and uses of the invention. Among others, theydid not incubate stem cell grafts with anti CD4 antibodies, but isolatedCD4+ cells separated out of spleens (murine) and buffy coats (human).

In addition, WO 2004/112835 describes, among others, methods involvingthe use of antibodies including antibodies directed against CD4. Here,anti CD4 antibodies were used to generate regulatory T cells over a longperiod in order to induce immunological tolerance.

In view of the above, there is still a need of promising alternative andimproved, respectively, therapeutic approaches that may lackdisadvantages of the prior art methodologies.

Furthermore, there are many instances whereby the efficacy of atherapeutic protein is limited by an unwanted immune reaction to thetherapeutic protein. Several mouse monoclonal antibodies have shownpromise as therapies in a number of human disease settings but incertain cases have failed due to the induction of significant degrees ofa human anti-murine antibody (HAMA) response (Schroff et al. (1985)).For monoclonal antibodies, a number of techniques have been developed inattempt to reduce the HAMA response (see e.g. WOA9106667). Theserecombinant DNA approaches have generally reduced the mouse geneticinformation in the final antibody construct whilst increasing the humangenetic information in the final construct. Notwithstanding, theresultant “humanized” antibodies have, in several cases, still elicitedan immune response in patients (Isaacs J. D. (1990)).

Antibodies are not the only class of polypeptide molecule administeredas a therapeutic agent against which an immune response may be mounted.Even proteins of human origin and with the same amino acid sequences asoccur within humans can still induce an immune response in humans.

Key to the induction of an immune response is the presence of peptideswithin the protein that can stimulate the activity of T cells viapresentation on MHC class II molecules, so-called “T-cell epitopes.”

MHC Class II molecules are a group of highly polymorphic proteins whichplay a central role in helper T cell selection and activation. The humanleukocyte antigen group DR (HLA-DR) are the predominant isotype of thisgroup of proteins; however, isotypes HLA-DQ and HLA-DP perform similarfunctions. In the human population, individuals bear two to four DRalleles, two DQ and two DP alleles. The structure of a number of DRmolecules have been solved and these appear as an open-ended peptidebinding groove with a number of pockets that engage amino acid sidechains (pocket residues) of the peptide (Stern et al. (1994)).Polymorphisms identifying the different allotypes of class II moleculecontributes to a wide diversity of different binding surfaces forpeptides within the peptide binding groove and, at the population level,ensures maximal flexibility with regard to the ability to recognizeforeign proteins and mount an immune response to pathogenic organisms.

An immune response to a therapeutic protein proceeds via the MHC classII peptide presentation pathway. Here exogenous proteins are engulfed byantigen presenting cells (APCs) and processed for presentation at thecell surface in association with MHC class II molecules of the DR, DQ orDP type. MHC Class II molecules are expressed by professional antigenpresenting cells, such as macrophages and dendritic cells amongstothers. Engagement of a MHC class II peptide complex by a cognate T cellreceptor on the surface of the T cell, together with the cross-bindingof certain other co-receptors such as the CD4 molecule, can induce anactivated state within the T cell. Activation leads to the release ofcytokines further activating other lymphocytes such as B cells toproduce antibodies or activating T killer cells as a full cellularimmune response.

T cell epitope identification is the first step to epitope eliminationas recognized in WO98/52976 and WO00/34317. In these teachings,predicted T cell epitopes are removed by the use of judicious amino acidsubstitutions within the protein of interest. Besides computationaltechniques, there are in vitro methods for measuring the ability ofsynthetic peptides to bind MHC class II molecules. An exemplary methoduses B-cell lines of defined MHC allotype as a source of MHC class IIbinding surface and may be applied to MHC class II ligand identification(Marshall et al. (1994); O'Sullivan et al. (1990); Robadey et al.(1997)). However, such techniques are not adapted for the screening ofmultiple potential epitopes to a wide diversity of MHC allotypes, norcan they confirm the ability of a binding peptide to function as a Tcell epitope.

Recently, techniques exploiting soluble complexes of recombinant MHCmolecules in combination with synthetic peptides have come into use(Kern et al. (1998); Kwok et al (2001)). These reagents and proceduresare used to identify the presence of T cell clones from peripheral bloodsamples from human or experimental animal subjects that are able to bindparticular MHC-peptide complexes and are not adapted for the screeningmultiple potential epitopes to a wide diversity of MHC allotypes.

CD4 is a surface glycoprotein primarily expressed on cells of the Tlymphocyte lineage including a majority of thymocytes and a subset ofperipheral T cells. Low levels of CD4 are also expressed by somenon-lymphoid cells although the functional significance of suchdivergent cellular distribution is unknown. On mature T cells, CD4serves a co-recognition function through interaction with MHC Class IImolecules expressed in antigen presenting cells. CD4+ T cells constituteprimarily the helper subset which regulates T and B cell functionsduring T-dependent responses to viral, bacterial, fungal and parasiticinfections.

During the pathogenesis of autoimmune diseases, in particular whentolerance to self antigens breaks down, CD4+ T cells contribute toinflammatory responses which result in joint and tissue destruction.These processes are facilitated by the recruitment of inflammatory cellsof the hematopoietic lineage, production of antibodies, inflammatorycytokines and mediators, and by the activation of killer cells.

CD4 antibodies are known in the art. An exemplary CD4 antibody,monoclonal mouse anti human CD4-antibody 30F16H5, is disclosed in DE3919294. Said antibody is obtainable from the hybridoma cell line ECACC88050502.

To reduce the immunogenicity of mouse anti-CD4 antibodies, humanizedanti-CD4 antibody have been previously engineered by cloning thehypervariable regions of a mouse antibody into frameworks provided byhuman immunoglobulins (e.g. Boon et al. (2002)). Although reducing theimmunogenicity compared to mouse anti-CD4, these humanized antibodystill elicited immune responses in several cases.

Furthermore, it is known from the art that such a “humanization” ofantibodies often results in antibodies with lower or significantly loweraffinity to the given target.

It is, hence, a further objective of the invention to provide formodified forms of an anti human CD4-antibody to reduce the immunereaction to mouse anti-CD4 antibodies. In particular, it is desirable toprovide anti-CD4 antibodies with a reduced number of T cell epitopeswhich may result in a reduced or absent potential to induce an immuneresponse in a human subject. Such proteins may be expected to display anincreased circulation time within a human subject capable of mounting animmune response to the non-modified antibody and may be of particularbenefit in chronic or recurring disease settings such as is the case fora number of indications for anti-CD4. While others have providedanti-CD4 antibody molecules including “humanized” forms, none of theseteachings recognize the importance of T cell epitopes to the immunogenicproperties of the protein nor have been conceived to directly influencesaid properties in a specific and controlled way according to the schemeof the present invention.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to an in vitro method ofmodifying a cell graft containing immune cells comprising the steps ofa) incubating a cell graft containing immune cells with an anti CD4antibody wherein said incubating is carried out for from 1 minute to 7days, b) removing unbound antibody from said graft.

In another aspect, the present invention relates to a modified cellgraft containing immune cells wherein said graft i) is obtainable inaccordance with the in vitro method of the invention; and/or ii)comprises anti CD4 antibodies bound to from 40% to 100% of theaccessible CD4 epitopes of said graft.

In another aspect, the present invention relates to the modified cellgraft containing immune cells of the invention for use in medicine,particularly for use in a method of treating in a subject one or morediseases treatable by transplantation.

In another aspect, the present invention relates to the use of an antiCD4 antibody for the in vitro modification of a cell graft containingimmune cells, the modification comprising incubating said graft withsaid antibody for from 1 minute to 7 days.

In other aspects, the invention relates to methods, uses and grafts asdefined in the claims and hereinbelow. In other aspects, the inventionrelates to particular antibodies disclosed herein.

A so-called additional aspect of the invention is summarized as follows:One facet of this additional aspect of the present invention relates toan anti human CD4-antibody comprising a heavy chain immunoglobulinvariable domain (VH) and a light chain immunoglobulin variable domain(VL), wherein at least one T cell epitope located outside the CDRs ofsaid immunoglobulin variable domains is removed from said immunoglobulinvariable domains, particularly to an anti human CD4-antibody as definedhereinbelow. In a preferred embodiment of the said additional aspect ofthe present invention, said antibody has the CDRs of the antibodyproduced by the hybridoma cell line ECACC 88050502, or said antibody hasthe CDRs of SEQ ID NO: 2 and SEQ ID NO: 12. In a particular embodiment,the heavy chain immunoglobulin variable domain comprises a sequenceselected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10; andthe light chain immunoglobulin variable domain comprises a sequenceselected from the group consisting of SEQ ID NO: 14, 16, 18, and 20. Inanother facet, the said additional aspect of the present inventionrelates to a pharmaceutical composition comprising said antibody and apharmaceutically acceptable carrier. In another facet, the saidadditional aspect of the present invention also relates to the use ofsaid antibody for the manufacture of a medicament for therapeuticallytreating a subject and methods of treatment using said antibody. Inanother facet, the said additional aspect of the present inventionrelates to a nucleic acid encoding a heavy chain and/or a light chainimmunoglobulin variable domain of said antibody, and to a vectorcomprising said nucleic acid, particularly wherein the nucleic acid isoperably linked to an expression control sequence. The said additionalaspect of the present invention further relates to a host cellcomprising said nucleic acid and/or at least one vector described above,as well as to a method of preparing an antibody of the said additionalaspect of the present invention, comprising culturing the host celldescribed above under conditions permitting expression under the controlof suitable expression control sequence(s), and purifying said antibodyfrom the medium of the cell. The said additional aspect of the presentinvention also relates to an anti human CD4-antibody, wherein theantibody is obtained using the expression vectors pANTVhG4 and pANTVκ.Generally, it is envisaged that the definitions, facets and embodimentsdescribed in context with the said additional aspect may also be appliedto the invention in general. Preferably, in said additional aspect, thespecificity and mode of action of the anti-CD4 antibodies are notaffected by the modification(s)

SHORT DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates the principle of antibody incubation ofgrafts as well as subsequent transplantation into a subject, such as ahuman being.

FIG. 2 contains an explanation of triple transgenic mice (TTG) as donorson a stable C57Bl/6 background. TTG mice express human CD4 and HLA-DRwhile murine CD4 molecules are knocked out. That allows thedetermination of specific human surface molecules after transplantation(unique analysis of chimerism) and the direct testing of anti human CD4antibodies in mice.

FIG. 3 shows an explanation of the experimental design. Bone marrowcells and splenocytes were taken from TTG mice, mixed and transplantedin lethally irradiated TTG mice with or without pre-treatment of antihuman CD4 antibodies. These experiments were compared by using donorcells from C57Bl/6 wild-type mice. Therapeutic effects (survival, organrepair, chimerism, GvHD) after transplantation were investigated.

treated cells, the survival rate was significantly increased (0% to 83%,p<0.001). The observed effect was specific for transplantation from TTGin Balb/c mice, because by using wild-type C57B1/6 mice as donors, theeffect could not be repeated. This result shows the specific binding ofthe antibody to human CD4 and thus wild-type donor cells are notaffected.

FIGS. 5A, 5B, and 5C illustrates the hematopoietic recovery aftertransplantation of BM/splenocytes from TTG mice in Balb/c mice with(FIG. 5A and FIG. 5C) or without (FIG. 5A and FIG. 5B) pre-incubation ofanti CD4 antibodies. In mice receiving pre-treated cells, thehematopoietic system recovered to the initial values aftertransplantation. Compared to recipients transplanted withoutpre-incubated cells, the reconstitution of monocytes and granulocyteswas before lymphocyte reconstitution (FIG. 5C).

FIG. 6 depicts the GvHD score (that includes body weight according toCooke et al., 1996) after transplantation of BM/splenocytes from TTGmice in Balb/c mice with or without pre-incubation of anti CD4antibodies. In mice receiving pre-treated cells, the GvHD score inantibody receiving mice was lower than in animals receiving bone marrowand splenocytes without pre-treatment of anti CD4 antibodies indicatingno GvHD development. Engraftment was also confirmed byimmunohistological analyses. In bone marrow cavities there was aprevalent form of hematopoiesis in transplanted animals and a stableengraftment of human CD4 expressing T cells.

FIG. 7 relates to experiments involving the engraftment of human CD4,murine CD4, and murine CD8 after transplantation of BM/splenocytes fromTTG mice in Balb/c mice without pre-incubation of anti human CD4antibodies. Twelve days after transplantation, human CD4, murine CD4,and murine CD8 cells could be stably detected and mice develop a severeGvHD.

FIG. 8 relates to experiments involving the engraftment of human HLA-DR3and murine MHC class I of TTG/C57B1/6 of (H-2Kb) after transplantationof BM/splenocytes from TTG mice in Balb/c mice without pre-incubation ofanti human CD4 antibodies. Twelve days after transplantation, humanHLA-DR3 and MHC class I of TTG/C57Bl/6 of H-2Kb and mice develop asevere GvHD.

FIG. 9 relates to experiments involving the engraftment of human CD4,murine CD8 and decrease of murine CD4 of TTG/C57Bl/6 of (H-2Kb) aftertransplantation of BM/splenocytes from TTG mice in Balb/c mice withpre-incubation of anti human CD4 antibodies. After transplantation astable engraftment could be observed without development of GvHD.

FIG. 10 depicts the survival rate after transplantation ofBM/splenocytes from TTG mice in Balb/c mice with or withoutpre-incubation of anti CD4 antibodies. Using an IgG1-isotype controlantibody, the preventive GvHD effect could not be observed.

FIG. 11 depicts exemplified vectors for expression of modified light andheavy chains in mammalian cells. dhfr is dihydrofolate reductase geneused for gene amplification by exposure of cells to increasingconcentrations of methotrexate; CMV P is the CMV IE promoter.

FIGS. 12A, 12B, 12C, 12D, and 12E depicts the DNA and amino acidsequences of exemplary modified heavy chain variable regions.

FIGS. 13A, 13B, 13C, 13D, and 13E depicts the DNA and amino acidsequences of exemplary modified light chain variable regions.

FIG. 14 depicts the relative binding of chimeric anti human CD4-antibodycompared to the parental mouse anti-CD4 antibody.

FIGS. 15A and 15B depicts the relative binding of exemplary modifiedanti-CD4 antibodies compared to the parental mouse anti-CD4 antibody andchimeric anti-CD4 antibody.

FIG. 16 depicts the relative binding of exemplary modified anti-CD4antibodies compared to the parental mouse anti-CD4 antibody and chimericanti-CD4 antibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention e.g. relates to the in vitro treatment of cellgrafts containing immune cells with antibodies, which avoids theirdirect application in vivo. That is, the present inventors could, forexample, show that the (preferably short term) incubation of bone marrowgrafts with an anti human CD4 antibody before transplantation of thesecell grafts containing immune cells prevents the development of GvHDafter transplantation as compared to isotype or untreated controls.

As opposed to work of the prior art, the presented work e.g. deals withthe short term incubation of a (stem cell) graft such as cellsuspensions containing T cells, in particular CD4 cells, with the aim oftolerance induction or immunosuppression to prevent e.g.Graft-versus-Host-Disease (GvHD).

Without intending to be bound by theory, the present inventors considerthe anti CD4 antibody incubation of (stem cell) grafts comprising CD4positive (immune) cells and subsequent removing of unbound antibodies toresult in a modified graft, wherein the antibody labeled cells areselectively inactivated by the antibody or are prepared for becominginactivated or becoming regulatory cells as soon as they encounterspecific antigen, such that e.g. GvHD is not initiated. It is assumedthat the anti CD4 antibody binds to immune cells (such as lymphocytes)bearing CD4 and thereby exerts its beneficial effect. In addition, it isconsidered feasible that the preferred anti CD4 antibodies describedherein show particularly advantageous features due to their binding of(a) specific epitope(s) in order to e.g. prepare the cell for subsequentinactivation.

As will be readily apparent to the skilled person, substantiallyreducing or avoiding the administration of free anti CD4 antibodies,i.e. anti CD4 antibodies that are not bound to an antigen located on thegraft, is advantageous. Generally, the present invention is consideredto be related to one or more of the following advantages: i) no directapplications of the antibodies to the recipients are required; ii) ashort term incubation of the graft, such as cell suspensions, tissues,and organs containing T cells, in particular CD4 cells; iii) GvHDprevention after transplantation of the graft of the invention; iv)prevention of other immunological complications after transplantation ofthe graft of the invention (e.g. cytokine-release syndrome); v)reduction of costs due to the avoidance or reduction of conventionalimmunosuppressive drugs and a significantly reduced amount of antibodiesas compared to systemic application; vi) improvement of survival ofpatients receiving a transplantation of the graft of the invention; vii)facilitation of transplantation of grafts also for patients such asolder patients, which can not be transplanted with regular grafts due toexpected immunological complications; and viii) use of HLA mismatchdonors for transplantation or less good HLA matches than without theinvention.

In a first aspect, the present invention relates to an in vitro methodof modifying a cell graft containing immune cells comprising the stepsof a) incubating a cell graft containing immune cells with an anti CD4antibody, especially wherein said incubating is carried out for from 1minute to 7 days, b) removing unbound antibody from said graft.

Antibodies and also anti CD4 antibodies are generally well known in theart. As used herein, by “antibody” is meant inter alia a protein of theimmunoglobulin family that is capable of specifically combining,interacting or otherwise associating with an antigen, wherein saidcombining, interacting or otherwise associating (such as binding) of theantibody to the antigen is mediated by complementarity-determiningregions (CDRs). Similarly, term “antigen” is used herein to refer to asubstance that is capable of specifically combining, interacting orotherwise associating with said antibody. In the context of the anti CD4antibody of the present invention the antigen is meant to be CD4,particularly human CD4.

As used herein, the term “CDR” refers to the“complementarity-determining region” of an antibody, i.e. to one of thehypervariable regions within an immunoglobulin variable domaincontributing to the determination of antibody specificity. CDRs are wellknown to a person skilled in the art. Typically, both the heavy chainimmunoglobulin variable domain and the light chain immunoglobulinvariable domain contain three CDRs.

In the context of the present invention, the term “antibody” isconsidered to also relate to antibody fragments including for exampleFv, Fab, Fab′ and F(ab′)2 fragments. Such fragments may be prepared bystandard methods (for example; Coligan et al., 1991-1997, incorporatedherein by reference). The present invention also contemplates thevarious recombinant forms of antibody derived molecular species wellknown in the art. Such species include stabilized Fv fragments includingsingle chain Fv forms (e.g., scFv) comprising a peptide linker joiningthe VH and VL domains, or an Fv stabilized by interchain disulphidelinkage (dsFv) and which contain additional cysteine residues engineeredto facilitate the conjoining of the VH and VL domains. Equally, othercompositions are familiar in the art and could include species referredto as “minibodies”; and single variable domain “dAbs”. Other speciesstill may incorporate means for increasing the valency of the modifiedantibody V-region domain, i.e. species having multiple antigen bindingsites for example by the engineering of dimerisation domains (e.g.,“leucine zippers”) or also chemical modification strategies. Moreover,the term “antibody” also relates to multimers of scFv such as diabodies,triabodies or tetrabodies, tandabs, flexibodies, bispecific antibodies,and chimeric antibodies, all known in the art. As used herein,antibodies are considered to also include any bivalent or multivalentantibodies. They also include any antibody derivatives and any otherderivatives known to the skilled person.

In some embodiments, the antibody is a polyclonal antibody. In preferredembodiments, the antibody is a monoclonal antibody.

According to the invention, the term “anti CD4 antibody” refers to anantibody, which has the ability to bind to CD4. Preferably, the anti CD4antibody is an anti human CD4 antibody. “CD4” or “cluster ofdifferentiation 4” refers to a protein, more precisely a surfaceglycoprotein, well known to the person skilled in the art (cf Bowers etal., 1997). In the present context CD4 may also refer to a fragment offull-length CD4, or an otherwise modified form of CD4, provided that thefragment or otherwise modified form still functions as an antigen in thecontext of the antibody of the present invention.

Preferred anti CD4 antibodies are selected from the group consisting ofMax16H5, OKT4A, OKTcdr4a, cMT-412, YHB.46. Most preferably, saidantibody is Max16H5. Cells for the production of Max16H5 have beendeposited with the ECACC (European Collection of Cell Cultures) withaccession number ECACC 88050502. Said antibody is also disclosed in DE3919294, which is incorporated by reference herein. As used herein, theantibody “Max16H5” may also be referred to as “Max.16H5”, “MAX16H5” or“MAX.16H5”, or also “30F16H5” (wherein the latter name is also the nameof deposited cells producing said antibody). Max.16H5 may also beobtained from the cell line MAX.16H5/30F16H5.

A further preferred anti CD4 antibody for use in the invention is16H5.chimIgG4. As used herein, said antibody may also be referred to as“16H5.chim” or as “CD4.16H5.chimIgG4” (wherein the latter name is alsothe name of deposited cells producing said antibody). 16H5.chimIgG4 maybe obtained from the cell line CD4.16H5.chimIgG4.

In detail, certain preferred anti CD4 antibodies in the context with thepresent invention are e.g. obtainable from any of the following depositsof biological material:

-   -   deposit with the European Collection of Cell Cultures having the        accession number ECACC 88050502;    -   deposit “MAX.16H5/30F16H5”, deposited with the DSMZ on Dec. 2,        2011;    -   deposit “CD4.16H5.chimIgG4”, deposited with the DSMZ on Dec. 2,        2011.

All of these deposits involve cells or cell lines, respectively, fromwhich particular anti CD4 antibodies in the context with the presentinvention may be obtained. Deposit ECACC 88050502 is e.g. also describedin application DE 3919294.

In some embodiments of the method, modified graft, or modified graft foruse of the invention, said anti CD4 antibody i) is selected from thegroup consisting of Max16H5, OKT4A, OKTcdr4a, cMT-412, YHB.46,particularly wherein said anti CD4 antibody is Max16H5; and/or ii) isantibody 30F16H5; and/or iii) is obtainable from a cell line depositedwith accession number ECACC 88050502; and/or iv) is obtainable from acell line MAX.16H5/30F16H5 deposited with the DSMZ on Dec. 2, 2011;and/or v) is antibody 16H5.chimIgG4; and/or vi) is obtainable from acell line CD4.16H5.chimIgG4 deposited with the DSMZ on Dec. 2, 2011;and/or vii) is an antibody comprising the VH and the VK of antibody16H5.chimIgG4; and/or viii) is an antibody comprising a VH and a VK ofan antibody obtainable from a cell line CD4.16H5.chimIgG4 deposited withthe DSMZ on Dec. 2, 2011; and/or ix) is an antibody comprising anycombination of a VH disclosed in FIGS. 12A, 12B, 12C, 12D, and 12E andof a VK disclosed in FIGS. 13A, 13B, 13C, 13D, and 13E, particularlywherein said combination is selected from VH1/VK1, VH2/VK2, VH4/VK2 andVH4/VK4, especially wherein said combination is VH2/VK2.

In particular embodiments, the anti CD4 antibody used in the inventionis “MAX.16H5”. In particular embodiments, the anti CD4 antibody used inthe invention is an antibody obtainable from cells of ECACC 88050502. Inparticular embodiments, the anti CD4 antibody used in the invention isan antibody obtainable from a deposit of biological material made by theApplicants with the DSMZ on Dec. 2, 2011. In particular embodiments, theanti CD4 antibody used in the invention is an antibody obtainable fromcells deposited by the Applicants with the DSMZ on Dec. 2, 2011. Inparticular embodiments, the anti CD4 antibody used in the invention isan antibody obtainable from a deposit with the European Collection ofCell Cultures having the accession number ECACC 88050502. In particularembodiments, the anti CD4 antibody used in the invention is an antibodyobtainable from cells “MAX.16H5/30F16H5” deposited with the DSMZ on Dec.2, 2011. In particular embodiments, the anti CD4 antibody used in theinvention is an antibody obtainable from cells “CD4.16H5.chimIgG4”deposited with the DSMZ on Dec. 2, 2011. In some embodiments, the antiCD4 antibody used in the invention comprises VH1 of FIGS. 12A, 12B, 12C,12D, and 12E. In some embodiments, the anti CD4 antibody used in theinvention comprises VH2 of FIGS. 12A, 12B, 12C, 12D, and 12E. In someembodiments, the anti CD4 antibody used in the invention comprises VH4of FIGS. 12A, 12B, 12C, 12D, and 12E. In some embodiments, the anti CD4antibody used in the invention comprises VK1 of FIGS. 13A, 13B, 13C,13D, and 13E. In some embodiments, the anti CD4 antibody used in theinvention comprises VK2 of FIGS. 13A, 13B, 13C, 13D, and 13E. In someembodiments, the anti CD4 antibody used in the invention comprises VK4of FIGS. 13A, 13B, 13C, 13D, and 13E. In some embodiments, the anti CD4antibody used in the invention comprises SEQ ID NO: 10. In someembodiments, the anti CD4 antibody used in the invention comprises SEQID NO: 8. In some embodiments, the anti CD4 antibody used in theinvention comprises SEQ ID NO: 4. In some embodiments, the anti CD4antibody used in the invention comprises SEQ ID NO: 20. In someembodiments, the anti CD4 antibody used in the invention comprises SEQID NO: 18. In some embodiments, the anti CD4 antibody used in theinvention comprises SEQ ID NO: 14. In some embodiments, the anti CD4antibody used in the invention comprises VH1 of FIGS. 12A, 12B, 12C,12D, and 12E and/or VK1 of FIGS. 13A, 13B, 13C, 13D, and 13E. In someparticularly preferred embodiments, the anti CD4 antibody used in theinvention comprises VH2 of FIGS. 12A, 12B, 12C, 12D, and 12E and/or VK2of FIGS. 13A, 13B, 13C, 13D, and 13E. In some embodiments, the anti CD4antibody used in the invention comprises VH4 of FIGS. 12A, 12B, 12C,12D, and 12E and/or VK2 of FIGS. 13A, 13B, 13C, 13D, and 13E. In someembodiments, the anti CD4 antibody used in the invention comprises VH4of FIGS. 12A, 12B, 12C, 12D, and 12E and/or VK4 of FIGS. 13A, 13B, 13C,13D, and 13E. In some embodiments, the anti CD4 antibody used in theinvention comprises SEQ ID NO: 10 and/or SEQ ID NO: 20. In some someparticularly preferred embodiments, the anti CD4 antibody used in theinvention comprises SEQ ID NO: 8 and/or SEQ ID NO: 18. In someembodiments, the anti CD4 antibody used in the invention comprises SEQID NO: 4 and/or SEQ ID NO: 18. In some embodiments, the anti CD4antibody used in the invention comprises SEQ ID NO: 4 and/or SEQ ID NO:14.

In some embodiments, the anti CD4 antibody used in the inventioncomprises the VH and the VK of the antibody 16H5.chimIgG4. In someembodiments, the anti CD4 antibody used in the invention comprises theCDRs of SEQ ID NO: 2 and SEQ ID NO: 12. In other preferred embodiments,the anti CD4 antibody for use in the invention is an anti CD4 antibodyof or in accordance with the said additional aspect of the inventiondisclosed in detail hereinbelow. Preferably, said anti CD4 antibody isas described in the embodiments thereof, where preferred embodiments areparticularly preferred. In general, the anti CD4 antibody used in theinvention may be any anti CD4 antibody disclosed herein.

In certain preferred embodiments, the anti CD4 antibody is selected fromantibodies recognizing the first and/or the second domain of the CD4molecules. In certain preferred embodiments, the anti CD4 antibody isselected from antibodies recognizing the same domain/s of the CD4molecules as Max 16H5.

As used herein, “unbound antibody” refers to an antibody which,following the step of incubating, is not bound to the graft. In otherwords, it refers to an antibody which is not essentially associated withits ligands on the graft.

As used herein, an “in vitro method” refers to a method that isperformed outside a living subject. It particularly also includes an “exvivo method”, such as in case of the graft comprising or being a tissueor an organ, but particularly excludes an “in vivo method” performedinside a living subject.

Preferably, according to the invention, the (step of) incubating iscarried out for a time sufficient to allow binding of said antibody tosaid graft. Preferably, said incubating is carried out for a timesufficient to allow the binding of anti CD4 antibodies to from 40% to100%, particularly 50% to 100%, particularly 60% to 100%, particularly70% to 100%, more particularly 80% to 100%, more particularly 90% to100%, more particularly 95% to 100%, more particularly 99% to 100%, ofthe accessible CD4 epitopes of said graft. Most preferably, followingsaid incubating, anti CD4 antibodies bind to essentially all of theaccessible CD4 epitopes of said graft.

An appropriate incubation period will easily be determined by the personskilled in the art. Usually, an appropriate incubation period willdepend on the type of graft used. A preferred incubation period may alsodependent on the amount of antibody used.

Generally, where the graft e.g. is a cell suspension, shorter incubationperiods will be required than where the graft e.g. is an organ.

Generally, where the graft comprises or is a tissue or an organ, longerincubation periods are preferred to allow the antibody to betransported—e.g. via diffusion—into the respective compartments.

Moreover, in any case, the skilled person may easily test the (status ofthe) binding of the anti CD4 antibodies according to methods well knownwithin the art that may, for example, involve flow cytometry.

Generally, short incubation periods are preferred over long incubationperiods in order to minimize any possible damage to the graft due to invitro processing.

According to the invention said incubating may e.g. be carried out forfrom 1 minute to 7 days. In some embodiments, said incubating is carriedout for from 1 to 150 minutes, particularly for from 10 minutes to 150minutes, more particularly for from 30 minutes to 150 minutes, moreparticularly for from 40 minutes to 120 minutes, more particularly forfrom 45 minutes to 90 minutes, especially for from 50 minutes to 70minutes. In other embodiments, said incubating is carried out for from150 minutes to 7 days, particularly for from 150 minutes to 5 days, moreparticularly from 150 minutes to 3 days, more particularly from 150minutes to 1 day, especially for from 150 minutes to 8 hours.

As to the removing of unbound (anti CD4) antibody in accordance with themethods and uses of the invention, various ways of performing said stepare known to the skilled person. One exemplary way of removing unboundantibody from the graft is by washing the graft. Washing may e.g. occurby employing centrifugation where the graft comprises or is a cellsuspension.

In the step of removing, preferably at least 40%, more particularly atleast 50%, more particularly at least 60%, more particularly at least70%, more particularly at least 80%, more particularly at least 90%, ofunbound (anti CD4) antibody are removed from the graft. Preferably, upto 100% of unbound (anti CD4) antibody are removed from the graft.

The amount of antibody employed in the above step of incubating is notparticularly limited. Appropriate amounts may easily be determined bythe person skilled in the art and may e.g. depend on the type of graftused. Preferably according to the invention, said incubating is carriedout with an antibody amount of from 0.1 μg to 100 mg.

In some embodiments, particularly where the graft is a cell suspension,said incubating is carried out with an antibody concentration of from0.1 μg/ml cell suspension to 150 μg/ml cell suspension, particularlyfrom 7 μg/ml cell suspension to 100 μg/ml cell suspension, moreparticularly from 30 μg/ml cell suspension to 100 μg/ml cell suspension,especially from 40 μg to 60 μg/ml cell suspension.

In some embodiments, particularly where the graft is a tissue or wherethe graft is an organ, said incubating is carried out with an antibodyamount of from 0.1 mg to 10 mg, particularly from 1 mg to 10 mg, moreparticularly from 2 mg to 9 mg, more particularly from 3 mg to 8 mg,especially from 4 mg to 6 mg.

In some embodiments, particularly where the graft is a tissue or wherethe graft is an organ, said incubating is carried out with an antibodyconcentration in the incubation solution of from 0.1 mg/ml to 10 mg/ml,particularly from 1 mg/ml to 10 mg/ml, more particularly from 2 mg/ml to9 mg/ml, more particularly from 3 mg/ml to 8 mg/ml, especially from 4mg/ml to 6 mg/ml. Preferably, the specified volume includes the volumeof said tissue or organ as well as the volume of the(antibody-containing) solution, in which said tissue or organ isincubated.

In some embodiments, particularly where the graft is a tissue or wherethe graft is an organ, said incubating is carried out by incubating saidtissue or organ in a solution having an antibody concentration of from10 μg/ml to 150 μg/ml, particularly from 20 μg/ml to 100 μg/ml, moreparticularly from 30 μg/ml to 100 μg/ml, especially from 40 μg/ml to 60μg/ml. Preferably, the specified volume includes the volume of saidtissue or organ as well as the volume of the (antibody-containing)solution, in which said tissue or organ is incubated.

When incubating tissues and/or organs with an antibody-containingsolution, the skilled person will readily perform such incubation suchas by means of a suitable container.

The selection of suitable amounts of antibody is well within theexpertise of the skilled person. Generally, higher amounts orconcentrations, respectively, of antibody are preferred where the graftcomprises or is a tissue or an organ. Moreover, the selection of anexact amount or a concentration, respectively, of antibody used willalso depend on the size of such tissue or organ.

In preferred embodiments, the above in vitro method or use is forreducing the likelihood of any one of the group consisting of GvHD,donor graft rejection, and organ rejection; particularly of GvHD, upontransplantation of said graft. In preferred embodiments, the above invitro method or use is for achieving tolerance within the transplantedimmunocompetent cells against the recipient's tissue upontransplantation of said modified graft. In preferred embodiments, theabove in vitro method or use is for achieving tolerance or partialtolerance within the recipient's tissue against the modified graft upontransplantation of said modified graft. As used herein, a “partialtolerance” is a partial immunotolerance results in a reduced immuneresponse. In preferred embodiments, the above in vitro method or use isfor silencing cell activation within said graft.

Grafts including cell grafts containing immune cells are very well knownto the person skilled in the art. As used herein, a “cell graftcontaining immune cells” is a graft comprising immune cells. The cellgraft containing immune cells is not particularly limited.

According to the present invention, the graft may comprise a cellsuspension, a tissue and/or an organ. Preferably, the graft is a cellsuspension, a tissue and/or an organ. More preferably, the graft isselected from the group consisting of a cell suspension, a tissue and anorgan.

In addition, in some preferred embodiments of the invention, the graftcomprises stem cells. A graft comprising stem cells may also be referredto herein as a stem cell graft.

According to the present invention, the graft comprises cells bearingthe CD4 antigen. Preferably, the graft comprises immune cells,particularly immune cells bearing the CD4 antigen. Such cells are wellknown to the person skilled in the art. In certain preferredembodiments, these immune cells are CD4 positive T lymphocytes orprecursor cells thereof. In certain preferred embodiments, these immunecells include, but are not limited to T helper cells and cells belongingto the monocyte and macrophage lineage, such as monocytes andmacrophages. Another example for such cells are microglia.

In some embodiments, said graft comprises, preferably is, a tissue,preferably a stem-cell-containing tissue. According to the presentinvention, suitable tissues include, but are not limited to blood,muscle, adipose tissue, connective tissue, epithelium, embryonic, andcellular tissue.

In other embodiments, said graft comprises, preferably is, an organ,preferably a stem-cell-containing organ. Suitable organs include, butare not limited to skin, intestine, kidney, and liver. Preferably, saidorgan is an intestine.

In preferred embodiments, said graft comprises, preferably is, a cellsuspension, preferably a stem-cell-containing cell suspension. Suitablecell suspensions and methods for obtaining them are well known to theskilled person. For example, a cell suspension graft may be obtained bypuncture of bones comprising bone marrow, e. g. puncture of the iliaccrests or sterna or taken from stem cell niches throughout the wholebody, e.g. fat tissue, tooth root, root of a hair and any other sourcementioned above.

In preferred embodiments, the cell suspension, particularly thestem-cell-containing cell suspension, comprises bone marrow cells, nonadherent bone marrow cells, peripheral blood cells, cord blood cells,cells from Wharton's jelly, placenta-derived cells, hair-root-derivedcells, and/or fat-tissue-derived cells. In preferred embodiments, thecell suspension, particularly the stem-cell-containing cell suspension,comprises lymphocytes, monocytes and/or macrophages.

In certain preferred embodiments the graft, particularly the cellsuspension, comprises any of bone marrow stem cells, peripheral bloodstem cells, umbilical cord blood stem cells, adult stem cells of thebone marrow such as NA-BMCs, embryonic stem cells and/or reprogrammedadult stem cells (i.e. induced pluripotent cells).

In some particular embodiments, the graft does not consist of or doesnot comprise embryonic stem cells. In some particular embodiments, thegraft does not consist of or does not comprise totipotent stem cells.

In preferred embodiments, the graft is a bone marrow suspension,particularly comprising bone marrow stem cells. Generally, the graft,particularly the bone marrow suspension, may additionally comprise anyof stem cells comprised in blood cells, cord blood cells, donorlymphocytes, peripheral blood stem cells, adult stem cells of the bonemarrow, embryonic stem cells and/or reprogrammed adult stem cells (i.e.induced pluripotent cells).

The graft, particularly the bone marrow suspension, may additionallycomprise any of stem cells comprised in blood cells, cord blood cells,donor lymphocytes, peripheral blood stem cells, and/or adult stem cellsof the bone marrow.

Generally, it is intended that the cell suspension also includes anycell suspension that comprises (any combination of) stem cells,optionally along with any (combination of) other cells.

The graft may also be a combination of grafts, such as a combination ofone or more of the grafts referred to above, e.g. a combination of anorgan and a cell suspension.

In a further aspect, the invention relates to a modified cell graftcontaining immune cells obtainable in accordance with an in vitro methodof the invention.

Likewise, the invention relates to a modified cell graft containingimmune cells, wherein said graft comprises anti CD4 antibodies bound tofrom 40% to 100% of the accessible CD4 epitopes of said graft.Preferably, the modified cell graft containing immune cells comprisesanti CD4 antibodies bound to 50% to 100%, particularly 60% to 100%,particularly 70% to 100%, more particularly 80% to 100%, moreparticularly 90% to 100%, more particularly 95% to 100%, moreparticularly 99% to 100%, of the accessible CD4 epitopes of said graft.Most preferably, essentially all of the accessible CD4 epitopes of thecell graft containing immune cells are bound to anti CD4 antibodies.

In a further aspect, the invention relates to a modified graft of theinvention for use in medicine.

In a further aspect, the invention relates to a modified graft of theinvention for use in a method of treating in a subject one or morediseases treatable by transplantation.

The use of grafts including cell grafts containing immune cells intransplantation is well known in the art. The present invention providesa modified graft which is intended to avoid severe side effects whichare associated with transplantation, as known in the art. Therefore, themodified grafts of the invention are used as it is known for theunmodified grafts.

Preferably, said subject is a mammalian subject, particularly a human.Preferably, said one or more diseases treatable by transplantationis/are selected from the group consisting of acute myeloid leukemia(AML); acute lymphoid leukemia (ALL); chronic myeloid leukemia (CML);myelodysplastic syndrome (MDS)/myeloproliferative syndrome; malignlymphomas, particularly selected from Morbus Hodgkin, high gradeNon-Hodgkin Lymphoma (NHL), mantle cell lymphoma (MCL), low malign NHL,chronic lymphatic leukemia (CLL), multiple myeloma; severe aplasticanemia; thalassemia; sickle cell anemia; immunological defectsparticularly selected from severe combined immunodeficiency (SCID),Wiskott-Aldrich syndrome (WAS), and hemophagocytic lymphohistiocytosis(HLH); inborn errors of metabolism particularly selected from lysosomalstorage disorders and disorders of peroxisomal function; autoimmunediseases; rheumatologic diseases; and recidivisms of any of the above.

Even more preferably, said one or more diseases are one or morehematological malignancies especially selected from acute myeloidleukemia (AML); acute lymphoid leukemia (ALL); chronic myeloid leukemia(CML); myelodysplastic syndrome (MDS)/myeloproliferative syndrome;malign lymphomas, particularly selected from Morbus Hodgkin, high gradeNon-Hodgkin lymphoma (NHL), mantle cell lymphoma (MCL), low malignNon-Hodgkin lymphoma (NHL), chronic lymphatic leukemia (CLL), multiplemyeloma; severe aplastic anemia; thalassemia; and sickle cell anemia.

Generally herein, said one or more diseases also include recidivisms ofany of the above as well as any combination of diseases mentionedherein.

In the latter aspects, preferably, the graft is further defined asdescribed hereinabove in connection with the in vitro methods of theinvention. That is, the graft may preferably be selected from the groupconsisting of a cell suspension, a tissue and an organ. More preferably,said graft is selected from the group consisting of a cell suspensioncomprising bone marrow cells, non adherent bone marrow cells, peripheralblood cells, cord blood cells, cells from Wharton's jelly,placenta-derived cells, hair-root-derived cells, and/orfat-tissue-derived cells; a cell suspension comprising lymphocytes,monocytes and/or macrophages; a stem-cell-containing tissue; and astem-cell-containing organ.

In some embodiments the treatment implies a reduced likelihood ofdeveloping any one of the group consisting of GvHD, donor graftrejection, and organ rejection; particularly of GvHD, upontransplantation of said graft. In other embodiments, the treatmentimplies tolerance within the transplanted immunocompetent cells againstthe recipient's tissue upon transplantation of said modified graft. Inother embodiments, the treatment implies tolerance against the modifiedgraft upon transplantation of said modified graft. In other embodiments,the treatment implies tolerance or partial tolerance within therecipient's tissue against the modified graft upon transplantation ofsaid modified graft. In other embodiments, the treatment is forsilencing cell activation within said graft. In preferred embodiments,the treatment implies/is for any combination of the above.

The amount of cells contained in the graft is not particularly limited.Any person skilled in the art will easily be able to choose appropriateamounts of a graft and of cells of the graft for transplantation.Furthermore, suitable guidance is also available e.g. from the specificguidelines for transplantation developed by the “DeutscheBundesärztekammer”, e.g. for human hematopoietic stem cells in patients.

Preferably, in accordance with the invention, particularly in case ofthe graft being a cell suspension, an amount of from 2×10⁶ cells to2×10¹⁰ nucleated cells, particularly of from 4×10⁶ to 1×10⁹ nucleatedcells, more particularly of from 1×10⁷ to 1×10⁸ nucleated cells areadministered to said subject, preferably to the human subject.

Where the graft comprises or is a tissue or an organ, any suitableamounts of said tissue or organ may be administered to said subject. Aswill be understood by the skilled person, cell numbers in tissues ororgans are difficult to determine. Particularly for this reason, theamount of cells contained in the graft is not particularly limited.Appropriate amounts will easily be determined or selected, respectively,by the skilled person, e.g. taking into consideration the particulartype of subject, graft and/or disease to be treated. In case of organs,the administration of whole organs is preferred.

In the methods, modified grafts, and modified grafts for use of thepresent invention, the graft may additionally be incubated with solublebioactive molecules, particularly with agents promotingimmunosuppression, immunotolerance and/or formation of regulatory Tcells or with any combination of such agents. Such agents preferablysupport the features or advantages, respectively, of the presentmethods, uses, modified grafts, or modified grafts for use describedhereinabove, such as reducing the likelihood of any one of the groupconsisting of GvHD, donor graft rejection, and organ rejection;particularly of GvHD, upon transplantation of said graft or such asachieving tolerance upon transplantation of said modified graft. Suchagents particularly include cytokines. In preferred embodiments, suchagent(s) is/are selected from the group consisting of 11-2, TGF-β,rapamycin, retinoic acid, 4-1BB ligand, and anti-CD28 antibodies, or anycombination thereof.

Likewise, in the modified grafts for use of the present invention, thegraft may optionally be administered to the subject together with anymedicament or combination of medicaments. Said medicament(s) may beadministered prior to, together with and/or following transplantation.Suitable administration modes and routes are not particularly limitedand will easily be chosen by the skilled person. Preferably, suchmedicament(s) support the features or advantages, respectively, of thepresent methods, uses, modified grafts, or modified grafts for usedescribed hereinabove, such as reducing the likelihood of any one of thegroup consisting of GvHD, donor graft rejection, and organ rejection.Non-limiting examples for such medicaments include rapamycin andretinoic acid.

In a further aspect, the invention features a method of treating asubject in need of such treatment with a modified graft of theinvention, particularly a modified graft obtainable in accordance withthe in vitro method of the invention. In preferred embodiments, saidgraft, subject, treatment, and/or disease are as described hereinabove.

In a further aspect, the invention relates to the use of an anti CD4antibody for the in vitro modification of a graft, particularly a cellgraft containing immune cells, the modification comprising incubatingsaid graft with said antibody for from 1 minute to 7 days, especiallywherein the modification additionally comprises removing unboundantibody from said graft. In preferred embodiments, said use is furtherdefined as described herein for the methods of the invention.

In a further aspect, the invention relates to the use of a modifiedgraft of the invention for the manufacture of a medicament for thetreatment of one or more diseases treatable by transplantation in asubject. In preferred embodiments, said use is further defined asdescribed herein for the methods of the invention. In particular thegraft, subject, treatment, and/or disease are preferably as describedhereinabove.

In an even further aspect of the methods, uses, modified grafts, ormodified grafts for use of the present invention, the anti CD4 antibodyis replaced by any other CD4 ligand. Preferred CD4 ligands include, butare not limited to peptide ligands (including naturally occurringpeptide ligands and peptide constructs) as well as aptamers. Such CD4ligands are known in the art.

In an even further aspect, the graft referred to in the methods, uses,modified grafts, and modified grafts for use of the present invention,may or may not comprise stem cells. That is, according to the latteraspect, the cell graft containing immune cells may be replaced by anygraft and includes a graft comprising stem cells as well as a graft notcomprising stem cells. In other words, the graft of the invention orused in accordance with the invention may be any graft or may be a cellgraft containing immune cells. In even other words, in certainembodiments the graft of the invention or used in accordance with theinvention comprises stem cells, whereas in other embodiments, the graftof the invention or used in accordance with the invention does notcomprise stem cells. In certain embodiments, the graft does not compriseisolated CD4⁺ cells.

As further described in the examples, the present inventors e.g.employed CD4−/− C57Bl/6 mice transgenic for human CD4 and HLA-DR3(triple transgenic mice, TTG; cf. Laub et al., 2000). The TTG mice havea complete functional murine immune system but without murine CD4instead of human CD4 and where in addition to murine MHC-II the humanHLA-DR3 is present. In this setting, the bone marrow cells can be takenas TTG grafts and incubated with anti human CD4 antibodies beforetransplantation in Balb/c wild-type mice. In this full MHC class Imismatch transplantation model, GvHD induction is highly presumably anda challenge for the anti CD4 antibody.

The engraftment of TTG/C57B16 donor cells in Balb/c mice was confirmedby flow cytometry. Stable Engraftment of H-2Kd (C57Bl/6), human CD4,HLA-DR, and a decrease of murine CD4 after transplantation indicates afull donor (TTG) hematopoiesis, first observed 12 days aftertransplantation. GvHD was confirmed by survival analysis, scoring systemand histology. Severity of GvHD was higher by using TTG donor cells thanC57Bl/6 wild-type donor cells. Survival of GvHD mice treated with antiCD4 antibody was significantly increased from 0 to 83%. Without antibodytreatment, GvHD mice died within 19-35 days. Used anti CD4 antibodieseffectively suppress GvHD development after murine HSCT in a full MHCmismatch model (TTG mice in Balb/c mice). This unique transplantationmodel allows direct testing of anti human CD4 antibodies in mice by astable murine GvHD model using TTG mice as donors. There was noinduction of GvHD after anti human CD4 pre-treatment of bone marrowgrafts from TTG mice. These findings are considered relevant for therefinement of strategies for suppression of reactive T cell clones.

In an even further aspect, the invention relates to an anti CD4antibody, selected from the group consisting of i) antibody16H5.chimIgG4; ii) an antibody obtainable from a cell lineCD4.16H5.chimIgG4 deposited with the DSMZ on Dec. 2, 2011; iii) anantibody comprising the VH and the VK of antibody 16H5.chimIgG4; iv) anantibody comprising a VH and a VK of an antibody obtainable from a cellline CD4.16H5.chimIgG4 deposited with the DSMZ on Dec. 2, 2011; v) anantibody comprising a combination of a VH disclosed in FIG. 12A, 12B,12C, 12D, or 12E and of a VK disclosed in FIG. 13A, 13B, 13C, 13D, or13E, wherein said combination is selected from VH1/VK1, VH2/VK2, VH4/VK2and VH4/VK4, especially wherein said combination is VH2/VK2.

In particular embodiments of this even further aspect, the anti CD4antibody of the invention is “MAX.16H5”. In particular embodiments, theanti CD4 antibody of the invention is an antibody obtainable from adeposit of biological material made by the Applicants with the DSMZ onDec. 2, 2011. In particular embodiments, the anti CD4 antibody of theinvention is an antibody obtainable from cells deposited by theApplicants with the DSMZ on Dec. 2, 2011. In particular embodiments, theanti CD4 antibody of the invention is an antibody obtainable from cells“MAX.16H5/30F16H5” deposited with the DSMZ on Dec. 2, 2011. Inparticular embodiments, the anti CD4 antibody of the invention is anantibody obtainable from cells “CD4.16H5.chimIgG4” deposited with theDSMZ on Dec. 2, 2011. In some embodiments, the anti CD4 antibody of theinvention comprises VH1 of FIG. 12A, 12B, 12C, 12D, or 12E. In someembodiments, the anti CD4 antibody of the invention comprises VH2 ofFIG. 12A, 12B, 12C, 12D, or 12E. In some embodiments, the anti CD4antibody of the invention comprises VH4 of FIG. 12A, 12B, 12C, 12D, or12E. In some embodiments, the anti CD4 antibody of the inventioncomprises VK1 of FIG. 13A, 13B, 13C, 13D, or 13E. In some embodiments,the anti CD4 antibody of the invention comprises VK2 of FIG. 13A, 13B,13C, 13D, or 13E. In some embodiments, the anti CD4 antibody of theinvention comprises VK4 of FIG. 13A, 13B, 13C, 13D, or 13E. In someembodiments, the anti CD4 antibody of the invention comprises SEQ ID NO:10. In some embodiments, the anti CD4 antibody of the inventioncomprises SEQ ID NO: 8. In some embodiments, the anti CD4 antibody ofthe invention comprises SEQ ID NO: 4. In some embodiments, the anti CD4antibody of the invention comprises SEQ ID NO: 20. In some embodiments,the anti CD4 antibody of the invention comprises SEQ ID NO: 18. In someembodiments, the anti CD4 antibody of the invention comprises SEQ ID NO:14. In some embodiments, the anti CD4 antibody of the inventioncomprises VH1 of FIG. 12A, 12B, 12C, 12D, or 12E and/or VK1 of FIG. 13A,13B, 13C, 13D, or 13E. In some particularly preferred embodiments, theanti CD4 antibody of the invention comprises VH2 of FIG. 12A, 12B, 12C,12D, or 12E and/or VK2 of FIG. 13A, 13B, 13C, 13D, or 13E. In someembodiments, the anti CD4 antibody of the invention comprises VH4 ofFIG. 12A, 12B, 12C, 12D, or 12E and/or VK2 of FIG. 13A, 13B, 13C, 13D,or 13E. In some embodiments, the anti CD4 antibody of the inventioncomprises VH4 of FIG. 12A, 12B, 12C, 12D, or 12E and/or VK4 of FIG. 13A,13B, 13C, 13D, or 13E. In some embodiments, the anti CD4 antibody of theinvention comprises SEQ ID NO: 10 and/or SEQ ID NO: 20. In some someparticularly preferred embodiments, the anti CD4 antibody of theinvention comprises SEQ ID NO: 8 and/or SEQ ID NO: 18. In someembodiments, the anti CD4 antibody of the invention comprises SEQ ID NO:4 and/or SEQ ID NO: 18. In some embodiments, the anti CD4 antibody ofthe invention comprises SEQ ID NO: 4 and/or SEQ ID NO: 14. In someembodiments, the anti CD4 antibody of the invention comprises the VH andthe VK of the antibody 16H5.chimIgG4.

Generally, the invention also relates to embodiments, where the term“comprises” or an equivalent term is replaced by “has” or an equivalentterm. For example, the invention generally also relates to embodiments,where the term “comprising” or an equivalent term is replaced by“having” or an equivalent term.

In the following, an additional aspect of the invention is described indetail:

In one facet, the said additional aspect of the present inventionrelates to an anti human CD4-antibody comprising a heavy chainimmunoglobulin variable domain (VH) and a light chain immunoglobulinvariable domain (VL), wherein at least one T cell epitope locatedoutside the CDRs of said immunoglobulin variable domains is removed fromsaid immunoglobulin variable domains.

Such antibodies are less immunogenic than their parental antibodies and,therefore, less likely to stimulate or activate T cells and, hence, areless likely to cause an undesired T cell mediated immune responseagainst the antibody, e.g. in a human subject.

Moreover, advantageously, said antibodies substantially retain thecapability of the corresponding non-modified antibody to bind to humanCD4 and, preferably, further retain at least one of their advantageousfeatures.

As used in said additional aspect of the invention, by “antibody” ismeant inter alia a protein of the immunoglobulin family that is capableof combining, interacting or otherwise associating with an antigen.

In the context of said additional aspect of the present invention, theterm “antibody” is preferably considered to also relate to antibodyfragments including for example Fv, Fab, Fab′ and F(ab′)2 fragments.Such fragments may be prepared by standard methods The said additionalaspect of the present invention preferably also contemplates the variousrecombinant forms of antibody derived molecular species well known inthe art. Such species include stabilized Fv fragments including singlechain Fv forms (e.g., scFv) comprising a peptide linker joining the VHand VL domains, or an Fv stabilized by interchain di-sulphide linkage(dsFv) and which contain additional cysteine residues engineered tofacilitate the conjoining of the VH and VL domains. Equally, othercompositions are familiar in the art and could include species referredto as “minibodies”; and single variable domain “dAbs.” Other speciesstill may incorporate means for increasing the valency of the modifiedantibody V-region domain, i.e. species having multiple antigen bindingsites for example by the engineering of dimerisation domains (e.g.,“leucine zippers”) or also chemical modification strategies. Moreover,the term “antibody” preferably also relates to multimers of scFv such asdiabodies, triabodies or tetrabodies, tandabs, flexibodies, bispecificantibodies, and chimeric antibodies. According to the said additionalaspect of the present invention, the term “anti human CD4-antibody”preferably refers to an antibody as defined above, which has the abilityto bind to human CD4. Moreover, as used in said additional aspect of theinvention, the term “non-modified antibody” or “parental antibody”preferably refers to a corresponding anti human CD4-antibody wherein, asopposed to the antibodies of the said additional aspect of the presentinvention, no T cell epitope located outside the CDRs of saidimmunoglobulin variable domains is removed from the immunoglobulinvariable domains. The term “antigen” is preferably used in saidadditional aspect of the invention to refer to a substance that iscapable of interacting with the antibody. In the context of the antibodyof the said additional aspect of the present invention the antigen ispreferably meant to be CD4, particularly human CD4. “CD4” or “cluster ofdifferentiation 4” refers to a protein, more precisely a surfaceglycoprotein, well known to the person skilled in the art. In thepresent context CD4 may preferably also refer to a fragment offull-length CD4, or an otherwise modified form of CD4, provided that thefragment or otherwise modified form still functions as an antigen in thecontext of the antibody of the said additional aspect of the presentinvention. The term “immunoglobulin domain” is preferably used in saidadditional aspect of the invention to inter alia refer to a proteindomain comprising an immunoglobulin fold. Immunoglobulin domains andproteins are well-known in the art. The term “VH” is preferably used insaid additional aspect of the invention to refer to the heavy chainvariable region of the heavy chain of an antibody. The “heavy chainvariable region” is also referred to as “heavy chain immunoglobulinvariable domain”. Also these terms are well-known in the art. The term“VL” is preferably used in said additional aspect of the invention torefer to the light chain variable region of the light chain of anantibody. The “light chain variable region” is also referred to as“light chain immunoglobulin variable domain”. Also these terms arewell-known in the art.

Preferably, a heavy chain immunoglobulin variable domain and a lightchain immunoglobulin variable domain together provide the bindingsurface capable of interacting with the antigen.

As used in said additional aspect of the invention, VH preferably meansa polypeptide that is about 110 to 125 amino acid residues in length,the sequence of which corresponds to any of the specified VH chains insaid additional aspect of the invention which in combination with a VLare capable of binding CD4 antigen. Similarly, VL preferably means apolypeptide that is about 95-130 amino acid residues in length thesequence of which corresponds to any of the specified VL chains in saidadditional aspect of the invention which in combination with a VH arecapable of binding the CD4 antigen.

Full-length immunoglobulin heavy chains have preferably a molecularweight of about 50 to 70 kDa and are usually encoded by a VH gene at theN-terminus and one of the constant region genes (e.g., [gamma], [alpha]or [epsilon]) at the C-terminus. Similarly, full-length light chainshave preferably a molecular weight of about 25 kDa and are usuallyencoded by a V-region gene at the N-terminus and a [kappa] or [lambda]constant region gene at the C-terminus.

The light chain of an antibody may be a “lambda” (“λ”) type chain or a“kappa” (“κ”) type chain. Accordingly, the light chain variable regionmay be a “lambda” (“V1”, “Vλ”) type light chain variable region or a“kappa” (“Vk”, “Vκ”) type light chain variable region.

In common with numerous monoclonal antibodies the light chain preferablyis a “kappa” (“κ”) type chain. Accordingly, VL is preferably a Vk (“VK”,“Vκ”) type chain.

In the context of the said additional aspect of the present invention,there are provided a number of different heavy chain immunoglobulinvariable domain sequences and light chain immunoglobulin variable domainsequences. The present disclosure provides no limit to the possiblecombinations of said variable domain sequences that may be comprised ina complete antibody molecule.

Preferably, the antibody of the said additional aspect of the presentinvention comprises two identical heavy chain immunoglobulin variabledomains and two identical light chain immunoglobulin variable domains,wherein said heavy chain immunoglobulin variable domains and light chainimmunoglobulin variable domains are selected from the variable domainsdisclosed in said additional aspect of the invention.

According to the said additional aspect of the present invention, theterm “T cell epitope” preferably refers to a peptide sequence, which hasa potential to bind to MHC molecules, preferably MHC class II molecules.Such sequences, in complex with MHC class II, may stimulate T cellsand/or bind (without necessarily activating) T cells.

Preferably, the term “T cell epitope” refers to a peptide sequence,which, when bound to MHC class II molecules, can be recognized by a Tcell receptor (TCR), and which can, at least in principle, cause thestimulation or activation of the corresponding T cell by engaging theTCR to promote a T cell response.

In order to identify T cell epitopes of an antibody, any of the insilico methods or in vitro methods which are known to a person skilledin the art and/or which are described or referred to in the presentapplication, may be used.

Preferably, a T cell epitope consists of eight or more amino acids, morepreferably of eight to twenty amino acids, more preferably of eight toeleven amino acids, particularly preferred of nine amino acids.

The term “peptide” as used in said additional aspect of the invention,is preferably a compound that includes two or more amino acids, whichare linked together by a peptide bond. Some peptides may contain only afew amino acid units. In the art, short peptides, e.g. peptides havingless than ten amino acid units, are sometimes referred to as“oligopeptides” whereas peptides containing a larger number of aminoacid residues, e.g. 10 to 100 or more than 100, are usually referred toas “polypeptides”.

Throughout the said additional aspect of the present invention, a T cellepitope is preferably said to be “removed” when T cell mediated immuneresponse based on said epitope against the antibody is reduced or,preferably, eliminated.

Preferably, T cell mediated immune response against the antibody isreduced (preferably eliminated), when the potential of the T cellepitope to bind to MHC molecules, preferably MHC class II molecules, isreduced (preferably eliminated).

According to an exemplary method described in Example 2, a modified Tcell epitope may be tested in silico for binding MHC class II alleles.In this method, a T cell epitope is considered to be “removed”, when alower number or no MHC Class II alleles are predicted to bind to themodified T cell epitope (see Example 2).

Other methods to measure the reduction or elimination of T cell mediatedimmune response are well known to the person skilled in the art andinclude, for example, in vitro MHC class II binding assays, utilizingeither purified MHC class II molecules or homozygous immortalized B celllines, or ex vivo T cell proliferation assays.

The removal of a T cell epitope may result in a decreased, preferablyabsent, immunogenicity displayed by the antibody. The term“immunogenicity” inter alia relates to an ability to provoke, induce orotherwise facilitate a humoral and or T cell mediated response in a hostanimal, in particular where the host animal is a human, and/or anability to elicit a response in a suitable in vitro assay. For example,the immunogenicity is said to be reduced if it is reduced compared to acorresponding parental antibody, e.g., a non-modified rodent or chimeric(rodent V-regions; human constant regions) monoclonal antibody.

As used in said additional aspect of the invention, the term “CDR”preferably refers to the “complementarity-determining region” of anantibody, i.e. to one of the hypervariable regions within animmunoglobulin variable domain contributing to the determination ofantibody specificity. CDRs are well known to a person skilled in theart. Typically, both the heavy chain immunoglobulin variable domain andthe light chain immunoglobulin variable domain contain three CDRs.

The CDRs in immunoglobulin variable domains may for example beidentified and defined according to the methods developed by Kabat,which are well-known to a person skilled in the art. According to apreferred embodiment of the said additional aspect of the presentinvention, the CDRs are defined according to Kabat (Kabat et al. (1991).

As used in said additional aspect of the invention, a removed T cellepitope is preferably said to be “located outside the CDRs of theimmunoglobulin variable domains”, when the sequence of the T cellepitope which is to be removed does not overlap with any of the CDRs ofsaid immunoglobulin variable domains. Besides, a removed T cell epitopeis also said to be “located outside the CDRs of the immunoglobulinvariable domains” in a case, in which the sequence of the T cell epitopewhich is to be removed does overlap with any of the CDRs of saidimmunoglobulin variable domains, in which, however, all of thealterations which have been made to such T cell epitope have been madeoutside the CDRs of the immunoglobulin variable domains.

The anti human CD4-antibody of the said additional aspect of the presentinvention may be a polyclonal antibody or a monoclonal antibody.Preferably, the antibody is a monoclonal antibody.

According to a preferred embodiment, the antibody is derived from themonoclonal antibody produced by hybridoma cell line ECACC 88050502.

The antibody produced by the hybridoma cell line ECACC 88050502 is a CD4antibody, and more specifically a monoclonal mouse anti humanCD4-antibody, also referred to as 30F16H5, which is, for example,disclosed in DE 3919294. Said antibody is obtainable from the hybridomacell line which was deposited with the ECACC (accession number88050502).

An antibody of the said additional aspect of the present invention issaid to be “derived” from the monoclonal antibody produced by hybridomacell line ECACC 88050502, when it has been obtained by any suitablemethod known to the person skilled in the art using the sequence of themonoclonal antibody produced by hybridoma cell line ECACC 88050502, orby using the hybridoma cell line ECACC 88050502.

Preferably, the antibody has the CDRs of the antibody produced by thehybridoma cell line ECACC 88050502, or the antibody has the CDRs of SEQID NO: 2 and SEQ ID NO: 12.

The CDRs of SEQ ID NO: 2 and SEQ ID NO: 12 have the sequenceshighlighted in italics in FIG. 12A and FIG. 13A, which figures depictpreferred immunoglobulin variable domain sequences of the parental antihuman CD4-antibody 30F16H5 (SEQ ID NO: 2 and SEQ ID NO: 12). In each ofFIG. 12A and FIG. 13A three CDRs are shown. Here, the CDRs in FIG. 12Aare formed by amino acids 31-35, 50-66 and 99-109 of SEQ ID NO: 2 andthe CDRs in FIG. 13A are formed by amino acids 24-33, 50-55 and 88-96 ofSEQ ID NO: 12.

Antibodies having the CDRs of the antibody produced by the hybridomacell line ECACC 88050502, or of SEQ ID NO: 2 and SEQ ID NO: 12 have ahigh potential to bind the human CD4 with an affinity comparable to thatof the parental antibody 30F16H5.

In another preferred embodiment of the antibody of the said additionalaspect of the present invention, with the exception of the differencesdue to the removal of one or more T cell epitopes from saidimmunoglobulin variable domains, the heavy chain immunoglobulin variabledomain is identical to the heavy chain immunoglobulin variable domain ofthe antibody produced by the hybridoma cell line ECACC 88050502, orcomprises a sequence identical to SEQ ID NO: 2; and the light chainimmunoglobulin variable domain is identical to the light chainimmunoglobulin variable domain of the antibody produced by the hybridomacell line ECACC 88050502, or comprises a sequence identical to SEQ IDNO: 12.

As described in Example 2b, the current inventors have discovered andherein disclose T cell epitopes of the parental anti human CD4-antibody30F16H5, which regions are e.g. depicted in Table 5 and Table 6 below.

This T cell epitopes referred to in said additional aspect of theinvention as EH1 to EH10 (“T cell epitope of heavy chain variableregion” 1 to 10) are individually depicted in Table 5 (SEQ ID NOs:21-30) and the T cell epitopes referred to in said additional aspect ofthe invention as EL1 to EL11 EH10 (“T cell epitope of light chainvariable region” 1 to 11) are individually depicted in Table 6 (SEQ IDNOs: 31-41). Each of these T cell epitopes may also be described onbasis of their position on the sequences of the respective parentalvariable regions SEQ ID NO: 2 and SEQ ID NO: 12, respectively.

Accordingly, in a preferred embodiment of the said additional aspect ofthe present invention, the at least one T cell epitope is selected fromthe group consisting of the T cell epitopes of the heavy chainimmunoglobulin variable domain at position 4 to 12 of SEQ ID NO: 2(EH1), position 10 to 18 of SEQ ID NO: 2 (EH2), position 11 to 19 of SEQID NO: 2 (EH3), position 20 to 28 of SEQ ID NO: 2 (EH4), position 37 to45 of SEQ ID NO: 2 (EH5), position 70 to 78 of SEQ ID NO: 2 (EH6),position 73 to 81 of SEQ ID NO: 2 (EH7), position 83 to 91 of SEQ ID NO:2 (EH8), position 107 to 115 of SEQ ID NO: 2 (EH9), position 110 to 118of SEQ ID NO: 2 (EH10), and the T cell epitopes of the light chainimmunoglobulin variable domain at position 2 to 10 of SEQ ID NO: 12(EL1), position 3 to 11 of SEQ ID NO: 12 (EL2), position 10 to 18 of SEQID NO: 12 (EL3), position 11 to 19 of SEQ ID NO: 12 (EL4), position 45to 53 of SEQ ID NO: 12 (EL5), position 53 to 61 of SEQ ID NO: 12 (EL6),position 59 to 67 of SEQ ID NO: 12 (EL7), position 61 to 69 of SEQ IDNO: 12 (EL8), position 62 to 70 of SEQ ID NO: 12 (EL9), position 70 to78 of SEQ ID NO: 12 (EL10), and position 97 to 105 of SEQ ID NO: 12(EL11).

It is understood that under certain circumstances additional regions ofsequence to those disclosed in said additional aspect of the inventioncan become immunogenic epitopes, for example in the event of infectionwith a pathogen expressing a protein or peptide with a similar sequenceto that of the present case.

According to a preferred embodiment of the said additional aspect of thepresent invention, the at least one T cell epitope is removed byalteration of at least one amino acid residue.

In particular, as used in said additional aspect of the invention, the“alteration” or “modification” of the at least one amino acid residuepreferably may be any of the following

-   -   the substitution of at least one originally present amino acid        residue by other amino acid residue,    -   the addition of at least one amino acid residue;    -   the deletion of at least one originally present amino acid        residue;    -   the chemical modification of at least one amino acid residue;

or a combination thereof.

Reference to the term “alteration” of at least one amino acid residuealso includes a situation, wherein, if necessary, additionalalteration(s), usually by substitution, addition or deletion of specificamino acid(s), are effected within the same T cell epitope or elsewherein the antibody molecule to substantially retain the capability of thecorresponding non-modified antibody to bind to human CD4. Morepreferably, such additional alteration(s) may be effected in order toadditionally retain one or more of the advantageous features of theantibody.

Preferably, one or more alterations are effected at one or more residuesfrom any or all of EH1 to EH10 and/or EL1 to EL11, preferably any or allof EH1 to EH10 and EL1 to EL11.

Particularly preferred, the alteration(s) are effected at one ore moreamino acids commonly designated as “pocket residues”, since they areengaged by the pockets of the MHC binding grooves. As will be understoodby a person skilled in the art, said pocket residues constitute residueswhich are of particular relevance to immunogenicity and, hence, are morelikely to reduce or, preferably, eliminate T cell mediated immuneresponse against the antibody. Generally, it is particularly preferredto provide modified antibody molecules in which amino acid alteration isconducted within the most immunogenic regions of the parental antibody.

However, amino acid alterations, either singly within a given epitope orin combination within a single epitope may not only be made at positionsequating to pocket residues with respect to the MHC class II bindinggroove, but at any point within the peptide sequence. All suchalterations fall within the scope of the said additional aspect of thepresent invention.

Moreover, as will be clear to the person skilled in art, multiplealternative sets of alterations could be arrived at which achieve theremoving of epitopes. The resulting sequences would, however, remainbroadly homologous with the specific compositions disclosed in saidadditional aspect of the invention and therefore fall under the scope ofthe invention. It would be typical to arrive at sequences that werearound 70%, or around 90%, or around 95%, or around 99% or morehomologous with the present specified sequences over their leasthomologous region and yet remain operationally equivalent. Suchsequences would equally fall under the scope of the present.

Preferably, the alteration of the at least one amino acid residue is thesubstitution of one ore more amino acids.

Accordingly, in a preferred embodiment of the antibody of the saidadditional aspect of the present invention, at least one amino acidwithin the at least one T cell epitope is substituted by another aminoacid for removing the at least one T cell epitope.

It is understood that single amino acid substitutions within a given Tcell epitope is a preferred route by which the epitope may beeliminated. Besides, combinations of substitution within a singleepitope may be contemplated and for example can be particularlyappropriate where individually defined epitopes are in overlap with eachother.

In various embodiments, more than 2 amino acid substitutions, or morethan 3 amino acid substitutions, or more than 4 amino acidsubstitutions, or more than 5 amino acid substitutions, or more than 6amino acid substitutions, or more than 7 amino acid, or more than 8, ormore than 9, or more than 10, or more than 11 or more than 12substitutions are made in the heavy chain and/or the light chain. Insome embodiments, between 1 and 21, between 5 and 20, or between 7 and14, amino acid substitutions are made in the heavy and light chain.

In each of the T cell epitopes EH1 to EH10 and EL1 to EL11 referred toabove, 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions may be present,provided that at least one substitution is present and that the antibodyretains its ability to bind to human CD4.

Preferably, the number of substitutions is selected such, that thenumber of MHC II alleles predicted to bind or bound, respectively, issignificantly decreased. Preferably, said number is decreased to atleast 50%, more preferably 40%, more preferably 30%, more preferably20%, more preferably 10%, more preferably 5%, more preferably 2%, morepreferably 1% and most preferably 0% as compared to the number ofalleles bound when no substitutions are present.

As exemplified in Example 2b, in a particular assay, the “number of MHCII alleles bound”, is the number of MHC class II alleles among a givenpanel of MHC class II alleles examined in the assay (e.g. 34 MHC classII alleles), which are found to be binding peptides for a T cell epitopeat issue. Said number is said to be “decreased”, when the number isreduced for a modified T cell epitope when compared to the unmodified Tcell epitope of the parental antibody.

As disclosed herein, various modified anti human CD4-antibodies, inwhich one ore more T cell epitopes are removed, have been created bymeans of MHC class II epitope removal involving amino acid substitution.Examples of particularly useful substitutions in this respect areprovided in FIGS. 12A, 12B, 12C, 12D, and 12E and FIGS. 13A, 13B, 13C,13D, and 13E, disclosing particular individual substitutions, i.e. theindividual substitutions highlighted in FIG. 12E and FIG. 13E, which maybe made in SEQ ID NO: 2 (cf. FIG. 12A) or SEQ ID NO: 12 (cf. FIG. 13A),respectively. These substitutions are also depicted in Table 5 (relatingto the heavy chain immunoglobulin domain) and Table 6 (relating to thelight chain immunoglobulin domain).

Hence, according to a preferred embodiment of the said additional aspectof the present invention, the at least one substitution is selected fromthe group consisting of T9S, V10E, A12K, Q19K, S28T, K38R, R40A, L70I,A72R, V73D, S91T, T115L, L116V in SEQ ID NO: 2, and I10T, M11L, L46A,V59S, I62S, S69D, R76S, L105I in SEQ ID NO: 12.

In a particularly preferred embodiment, 0, 1, 2, or 3 substitutions arewithin each of EH1 and EH6; 0, 1 or 2 substitutions are within each ofEH2, EH5, and EH10; 0 or 1 substitutions are within each of EH3, EH4,EH7, EH8, and EH9; 0, 1 or 2 substitutions are within each of EL2, EL3,EL7, EL8, and EL9; and 0 or 1 substitutions are within each of EL1, EL4,EL5, EL6, EL10, and EL11, with the proviso that at least onesubstitution is present.

According to a embodiment of the said additional aspect of the presentinvention, 6, 8, or 10 T cell epitopes are removed from the heavy chainimmunoglobulin variable domain, and/or 5, 9, 10, or 11 T cell epitopesare removed from the light chain immunoglobulin variable domain of theantibody of the said additional aspect of the present invention.

More preferably, 6, 8, or 10 T cell epitopes are removed from the heavychain immunoglobulin variable domain, and 5, 9, 10, or 11 T cellepitopes are removed from the light chain immunoglobulin variable domainof the antibody of the said additional aspect of the present invention.

According to one embodiment, all T cell epitopes are removed from theimmunoglobulin variable domains.

Furthermore, according to the said additional aspect of the presentinvention, exemplary groups of substitutions have been made in theimmunoglobulin variable domains, which groups are comprised by SEQ IDNOs: 4, 6, 8, and 10 (depicted in FIGS. 12B, 12C, 12D, and 12E) and SEQID NOs: 14, 16, 18, and 20 (depicted in FIGS. 13B, 13C, 13D, and 13E).

Hence, further preferred embodiments of the said additional aspect ofthe present invention are anti human CD4-antibodies, wherein the heavychain immunoglobulin variable domain comprises a sequence selected fromthe group consisting of SEQ ID NOs: 4, 6, 8, and 10; and/or the lightchain immunoglobulin variable domain comprises a sequence selected fromthe group consisting of SEQ ID NOs: 14, 16, 18, and 20.

More preferably, the heavy chain variable domain comprises a sequenceselected from the group consisting of SEQ ID NOs: 4, 6, 8, and 10; andthe light chain variable domain comprises a sequence selected from thegroup consisting of SEQ ID NO: 14, 16, 18, and 20.

Even more preferred, the heavy chain immunoglobulin variable domaincomprises a sequence identical to SEQ ID NO: 4 and the light chainimmunoglobulin variable domain comprises a sequence identical to SEQ IDNO: 14; the heavy chain immunoglobulin variable domain comprises asequence identical to SEQ ID NO: 4 and the light chain immunoglobulinvariable domain comprises a sequence identical to SEQ ID NO: 20; theheavy chain immunoglobulin variable domain comprises a sequenceidentical to SEQ ID NO: 6 and the light chain immunoglobulin variabledomain comprises a sequence identical to SEQ ID NO: 14; the heavy chainimmunoglobulin variable domain comprises a sequence identical to SEQ IDNO: 6 and the light chain immunoglobulin variable domain comprises asequence identical to SEQ ID NO: 16; the heavy chain immunoglobulinvariable domain comprises a sequence identical to SEQ ID NO: 6 and thelight chain immunoglobulin variable domain comprises a sequenceidentical to SEQ ID NO: 20; the heavy chain immunoglobulin variabledomain comprises a sequence identical to SEQ ID NO: 8 and the lightchain immunoglobulin variable domain comprises a sequence identical toSEQ ID NO: 16; the heavy chain immunoglobulin variable domain comprisesa sequence identical to SEQ ID NO: 8 and the light chain immunoglobulinvariable domain comprises a sequence identical to SEQ ID NO: 20; theheavy chain immunoglobulin variable domain comprises a sequenceidentical to SEQ ID NO: 10 and the light chain immunoglobulin variabledomain comprises a sequence identical to SEQ ID NO: 14; the heavy chainimmunoglobulin variable domain comprises a sequence identical to SEQ IDNO: 10 and the light chain immunoglobulin variable domain comprises asequence identical to SEQ ID NO: 16; or the heavy chain immunoglobulinvariable domain comprises a sequence identical to SEQ ID NO: 10 and thelight chain immunoglobulin variable domain comprises a sequenceidentical to SEQ ID NO: 20.

As can be taken from Example 2, antibodies comprising these particularvariable domain sequence combinations show advantageous featuresparticularly as regards their binding affinity to the CD4 antigen.

Particularly preferred, the heavy chain immunoglobulin variable domaincomprises a sequence identical to SEQ ID NO: 4 and the light chainimmunoglobulin variable domain comprises a sequence identical to SEQ IDNO: 14; the heavy chain immunoglobulin variable domain comprises asequence identical to SEQ ID NO: 6 and the light chain immunoglobulinvariable domain comprises a sequence identical to SEQ ID NO: 16; theheavy chain immunoglobulin variable domain comprises a sequenceidentical to SEQ ID NO: 10 and the light chain immunoglobulin variabledomain comprises a sequence identical to SEQ ID NO: 16; or the heavychain immunoglobulin variable domain comprises a sequence identical toSEQ ID NO: 10 and the light chain immunoglobulin variable domaincomprises a sequence identical to SEQ ID NO: 20.

As shown in Example 2c disclosed herein, the antibodies according tothese embodiments show a binding to human CD4, which is improved whencompared to the parental monoclonal mouse anti CD4-antibody 30F16H5,which was used as a reference.

Generally, as indicated above, the modified antibody of the presentexhibits an ability to bind to human CD4. As used in said additionalaspect of the invention, an antibody is preferably said to“substantially retain” its capability to bind to human CD4, if theaffinity for its target antigen CD4 is at least 5%, more preferably atleast 10%, more preferably at least 20%, more preferably at least 40%,more preferably at least 50%, more preferably at least 60%, morepreferably at least 70%, more preferably at least 80%, more preferablyat least 90%, more preferably at least 100% of the affinity exhibited bythe non-modified monoclonal anti CD4-antibody.

Various methods for the measurement of the affinity of antibodies arewell known to a person skilled in the art. Suitable methods include themeasurement of the affinity via competition ELISA as described inExample 2c herein, or a Scatchard analysis or analysis using a Biacore(Perkin Elmer) or similar instrument.

In a preferred embodiment of the said additional aspect of the presentinvention, the affinity for its target antigen CD4 is within an order ofmagnitude higher or lower than the affinity exhibited by the parentalanti-CD4 monoclonal antibody.

For example, the binding affinity of the antibody of the said additionalaspect of the present invention to CD4 is preferably within one order ofmagnitude higher or lower than the binding affinity of the antibodyproduced by the hybridoma cell line ECACC 88050502.

More preferably, the binding affinity is twofold higher or lower thanthe binding affinity of the antibody produced by the hybridoma cell lineECACC 88050502.

According to particularly preferred embodiments, the antibody has ahigher binding affinity to CD4 than the antibody produced by thehybridoma cell line ECACC 88050502.

Preferably, the modified antibodies disclosed in said additional aspectof the invention in additionally retain at least one and most preferablyall of the functional activities of the parental anti humanCD4-antibody. Embodiments of the said additional aspect of the presentinvention therefore encompass modified antibodies in which one or more,and most preferably all of the beneficial technical features associatedwith the therapeutic efficacy of the parental non-modified antibody areexhibited, while the antibody has a reduced ability to bind to MHC classII molecules and/or induces a weaker or no immune response in a subject.

Preferably, the anti human CD4-antibody heavy chain further comprises ahuman IgG4 constant region domain and the light chain further comprisesa human kappa constant region domain. Accordingly, in another preferredembodiment, the heavy chain variable region of the antibody of the saidadditional aspect of the present invention is linked to a human IgG4constant region domain, and the light chain variable region of theantibody of the said additional aspect of the present invention islinked to a human kappa constant region domain. IgG4 has a lowpropensity to stimulate effector functions such as ADCC (antibodydependent cell-mediated cytotoxicity) and CDC (complement-induced celldeath) and cannot therefore stimulate a pro-inflammatory response in thepatient.

In particular embodiments, the anti human CD4-antibody further comprisesa human IgG4 constant region domain adjacent to a heavy chain variableregion sequence selected from SEQ ID NOs: 4, 6, 8, and 10 and a humankappa constant region domain adjacent to a light chain variable regionsequence selected from SEQ ID NOs: 14, 16, 18, 20.

As described hereinabove, these exemplary sequences for the heavy chainand light chain variable region, respectively, are preferred variableregion sequences.

According to another facet of the said additional aspect of the presentinvention, the antibody of the said additional aspect of the presentinvention is obtained using the expression vectors pANTVhG4 and pANTVκ.

As a non-limiting example, the antibody of the said additional aspect ofthe present invention can be obtained using the expression vectorspANTVhG4 and pANTVκ as described in Example 2. Besides, any othersuitable method(s) well known to a person skilled may be employed, inwhich modified antibodies are constructed on basis of particularantibody sequences such as the ones contained in the expression vectorspANTVhG4 and pANTVκ.

In another facet, the said additional aspect of the present inventionrelates to a method of preparing the anti human CD4-antibody of the saidadditional aspect of the present invention comprising the followingsteps:

-   -   (i) providing the amino acid sequence of the antibody derivable        from the hybridoma cell line ECACC 88050502 or part thereof;    -   (ii) identifying one or more T cell epitopes within the amino        acid sequence of the antibody or part thereof by any method        including determination of the binding of the peptides to MHC        molecules using in vitro or in silico techniques or biological        assays;    -   (iii) designing new sequence variants with one or more amino        acids within the identified T cell epitopes modified in such a        way to substantially reduce or eliminate binding of the peptides        to MHC molecules measured by in vitro or in silico techniques or        biological assays; and    -   (iv) constructing such sequence variants by recombinant DNA        techniques and testing said sequence variants in order to        identify one or more sequence variants having the properties of        the anti human CD4-antibody of the said additional aspect of the        present invention.

The identification of T cell epitopes according to step (ii) can becarried out according to methods described previously in the art.Suitable methods are e.g. disclosed in WO 98/59244; WO 00/34317; U.S.Application 20030153043, all incorporated herein by reference. In themethod described above, sequence variants are preferably created in sucha way to avoid creation of new T cell epitopes by the sequencevariations unless such new T cell epitopes are, in turn, modified insuch a way to substantially reduce or eliminate binding of peptides toMHC class II molecules. In practice, when conducting alterations to theprotein sequence, it is preferably avoided that the contemplated changesintroduce new immunogenic epitopes by re-testing the contemplatedsequence for the presence of epitopes and or of MHC class II ligands byany suitable means.

In various embodiments, the modified antibodies of the said additionalaspect of the present invention are generated by expression of differentcombinations of the VH and VL genes specified in said additional aspectof the invention. All such combinations of heavy and light chain areencompassed by the said additional aspect of the present invention.

Generally, constitution of the complete antibody molecule may beachieved by recombinant DNA techniques and methods for purifying andmanipulating antibody molecules well known in the art. Necessarytechniques are explained fully in standard literature, which iswell-known to the skilled person.

The preferred molecules of this said additional aspect of the presentinvention can be prepared in any of several ways but is most preferablyconducted exploiting routine recombinant methods. It is a relativelyfacile procedure to use the protein sequences and information providedin said additional aspect of the invention to deduce a polynucleotide(DNA) encoding any of the preferred antibody V-regions. This can beachieved for example using computer software tools such as the DNAstarsoftware suite [DNAstar Inc, Madison, Wis., USA] or similar. Any suchDNA sequence with the capability of encoding the preferred polypeptidesof the present or significant homologues thereof, should be consideredas embodiments of this said additional aspect of the present invention.

As a general scheme, any of the VH or VL chain genes can be made usinggene synthesis and cloned into a suitable expression vector. In turn theexpression vector is introduced into a host cell and cells selected andcultured. The antibody molecules are readily purified from the culturemedium and formulated into a preparation suitable for therapeuticadministration.

By way of a non-limiting example, one such scheme involves a genesynthesis process using panels of synthetic oligonucleotides. The genesare assembled using a ligase chain reaction (LCR) wherein theoligonucleotides featuring complementary ends are allowed to annealfollowed by amplification and fill-in using a polymerase chain reaction(PCR). The PCR is driven by addition of an increased concentration ofthe flanking oligonucleotides to act as primers. The PCR products areassembled into full-length antibody genes by further PCR from vectorscontaining 5′ and 3′ immunoglobulin gene flanking regions andsub-cloning into expression vectors for expression of whole antibody.The assembled VH and VL genes can serve as templates for mutagenesis andconstruction of multiple variant antibody sequences such as any of thosedisclosed in said additional aspect of the invention. It is particularlyconvenient to use the strategy of “overlap extension PCR” as describedby (Higuchi et al. (1998)), although other methodologies and systemscould be readily applied.

Full-length immunoglobulin genes containing the variable regioncassettes are most conveniently assembled using overlapping PCR andsub-cloned into expression vectors containing the desired immunoglobulinconstant region domains. The expression vectors may be introduced into amammalian or other host cell for example using electroporationtechniques. The NS0 cell line is a non-immunoglobulin producing mousemyeloma, obtained from the European Collection of Animal Cell Cultures(ECACC) and is particularly suitable example host cell line for thisprocedure. Cell lines secreting antibody are expanded and antibody canbe readily purified for example by use of protein A affinitychromatography (Harlow E & Lane D (2006)). The concentration of thepurified antibody can be determined using an enzyme linked immunosorbentassay (ELISA) detecting the human kappa constant region of theantibodies of interest.

In as far as the said additional aspect of the present invention relatesto modified anti-CD4 antibodies, compositions containing such modifiedantibodies or fragments of modified antibodies and related compositionsare also considered to be within the scope of the invention.

Therefore, the said additional aspect of the present invention furtherrelates to a pharmaceutical composition comprising the anti humanCD4-antibody of the said additional aspect of the present invention anda pharmaceutically acceptable carrier.

The therapeutic compositions of the anti human CD4-antibody of the saidadditional aspect of the present invention may be used in conjunctionwith a pharmaceutically acceptable excipient. The pharmaceuticalcompositions according to the said additional aspect of the presentinvention are prepared conventionally, comprising substances that arecustomarily used in pharmaceuticals, including excipients, carriers,adjuvants, and buffers. The compositions can be administered, e.g.,parenterally, enterally, intramuscularly, subcutaneously, intravenously,or other routes useful to achieve an effect. Conventional excipientsinclude pharmaceutically acceptable organic or inorganic carriersubstances suitable for parenteral, enteral, and other routes ofadministration that do not deleteriously react with the agents. Forparenteral application, particularly suitable are injectable sterilesolutions, preferably oil or aqueous solutions, as well as suspensions,emulsions or implants, including suppositories. Ampoules are convenientunit dosages. The pharmaceutical preparations can be sterilized and, ifdesired, mixed with stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, or other substances that do notreact deleteriously with the active compounds.

The modified antibodies disclosed in said additional aspect of theinvention are useful in a number of important diseases in man includingespecially autoimmune conditions including, but not limited to, multiplesclerosis, rheumatoid arthritis, systemic vasculitis, uveitis,inflammatory bowel disease and scleroderma and also for use intransplantations. Hence, the antibodies of the said additional aspect ofthe present invention can be used in therapeutic treatment. Non-limitingexamples encompass a method of treating autoimmune conditions in apatient comprising administering an effective amount of a modifiedantibody according to the said additional aspect of the presentinvention. In various embodiments the autoimmune condition is multiplesclerosis, rheumatoid arthritis, systemic vasculitis, uveitis,inflammatory bowel disease or scleroderma. Another example is a methodof immunosuppressing a patient prior to or subsequent to transplantationof an organ comprising administering to said patient an effective amountof an antibody according to the said additional aspect of the presentinvention. In one embodiment, the organ for transplantation is a renaltransplant. The said additional aspect of the present invention alsorelates to methods for therapeutic treatment of humans using themodified antibody compositions. For administration to an individual, anyof the modified antibody compositions would preferably be produced to beat least 80% pure and free of pyrogens and other contaminants.

Accordingly, in one further facet, the said additional aspect of thepresent invention relates to a method of therapeutic treatmentcomprising administering the antibody to a subject, preferably to apatient. Preferably, the method is for treating an autoimmune condition,particularly an autoimmune condition selected from multiple sclerosis,rheumatoid arthritis, systemic vasculitis, uveitis, inflammatory boweldisease and scleroderma. According to another preferred embodiment, themethod is for immunosuppressing a patient prior to or subsequent totransplantation of an organ, particularly a kidney.

Preferably, the subject is a human. Preferably, an effective amount ofthe antibody is administered.

In a related facet, the said additional aspect of the present inventionrelates to the use of the anti human CD4-antibody of the said additionalaspect of the present invention for the manufacture of a medicament fortherapeutically treating a subject. Preferably, the medicament is fortreating an autoimmune condition, particularly an autoimmune conditionselected from multiple sclerosis, rheumatoid arthritis, systemicvasculitis, uveitis, inflammatory bowel disease and scleroderma.According to another preferred embodiment, the medicament is forimmunosuppressing a patient prior to or subsequent to transplantation ofan organ, particularly a kidney. Preferably, the subject is a human.

In another related facet, the said additional aspect of the presentinvention relates to the antibody of the said additional aspect of thepresent invention for use in a method of therapeutic treatment.Preferably, the method is for treating an autoimmune condition,particularly an autoimmune condition selected from multiple sclerosis,rheumatoid arthritis, systemic vasculitis, uveitis, inflammatory boweldisease and scleroderma. According to another preferred embodiment, themethod is for immunosuppressing a patient prior to or subsequent totransplantation of an organ, particularly a kidney. Preferably, thesubject is a human.

In the methods of treatments and medical uses of the said additionalaspect of the present invention, the actual dosage of the anti-CD4antibodies of the said additional aspect of the present inventionemployed will depend on a variety of factors including the type andseverity of disorder being treated, and other treatment modality ormodalities selected. Guidance for dosage regimens is obtained fromdosing of humanized anti-CD4 known in the art.

In a still other facet, the said additional aspect of the presentinvention relates to a nucleic acid encoding a heavy chain and/or alight chain immunoglobulin variable domain of the anti humanCD4-antibody of the said additional aspect of the present invention.Preferably, the nucleic acid comprises a sequence selected from thegroup consisting of SEQ ID NOs: 3, 5, 7, 9, 13, 15, 17, and 19.

Also part of the said additional aspect of the present invention arenucleic acids nucleic acid encoding a heavy chain and/or a light chainimmunoglobulin variable domain of the anti human CD4-antibody of thesaid additional aspect of the present invention, which differ from SEQID NOs: 3, 5, 7, 9, 13, 15, 17, and 19 due to the degeneracy of thegenetic code.

Degeneracy in relation to polynucleotides refers to the fact wellrecognized in the art that in the genetic code many amino acids arespecified by more than one codon. The degeneracy of the code accountsfor 20 different amino acids encoded by 64 possible triplet sequences ofthe four different bases.

In another facet, the said additional aspect of the present inventionrelates to a vector comprising a nucleic acid as described above.Preferably, the nucleic acid is operably linked to an expression controlsequence.

In some embodiments, the expression vector comprises a nucleic acidsequence encoding a V-region heavy or light chain comprising a modifiedsubstituted variant of SEQ ID NO: 2 or SEQ ID NO: 12 with a reducednumber of T cell epitopes, operably linked to an expression controlsequence. In various embodiments, the expression vector comprises or isderived from the pANTVhG4 vector (for VH) and the pANTVκ vector for VLas depicted in FIG. 11.

In another facet, the said additional aspect of the present inventionrelates to a host cell comprising a nucleic acid as described aboveand/or at least one vector as described above. Preferably, the host cellcomprises one or more vectors which each comprise a nucleic acid asdescribed above. Preferably the host cell comprises two vectors whicheach comprise a nucleic acid as described above.

The said additional aspect of the present invention further relates to amethod of preparing the anti human CD4-antibody of the said additionalaspect of the present invention comprising culturing the host celldescribed above under conditions permitting expression under the controlof suitable expression control sequence(s), and purifying said antibodyfrom the medium of the cell.

In the following items 1 to 33 certain embodiments of the saidadditional aspect of the present invention are described:

-   1. An anti human CD4-antibody comprising a heavy chain    immunoglobulin variable domain (VH) and a light chain immunoglobulin    variable domain (VL), wherein at least one T cell epitope located    outside the CDRs of said immunoglobulin variable domains is removed    from said immunoglobulin variable domains.-   2. The antibody of item 1, wherein the antibody is a monoclonal    antibody.-   3. The antibody of item 2, wherein the antibody is derived from the    monoclonal antibody produced by hybridoma cell line ECACC 88050502.-   4. The antibody of any one of items 1 to 3, wherein the antibody has    the CDRs of the antibody produced by the hybridoma cell line ECACC    88050502, or wherein the antibody has the CDRs of SEQ ID NO: 2 and    SEQ ID NO: 12.-   5. The antibody of any one of items 1 to 4, wherein, with the    exception of the differences due to the removal of one or more T    cell epitopes from said immunoglobulin variable domains,    -   the heavy chain immunoglobulin variable domain is identical to        the heavy chain immunoglobulin variable domain of the antibody        produced by the hybridoma cell line ECACC 88050502, or comprises        a sequence identical to SEQ ID NO: 2; and    -   the light chain immunoglobulin variable domain is identical to        the light chain immunoglobulin variable domain of the antibody        produced by the hybridoma cell line ECACC 88050502, or comprises        a sequence identical to SEQ ID NO: 12.-   6. The antibody of any one of items 1 to 5, wherein the at least one    T cell epitope is selected from the group consisting of    -   the T cell epitopes of the heavy chain immunoglobulin variable        domain at position 4 to 12 of SEQ ID NO: 2 (EH1), position 10 to        18 of SEQ ID NO: 2 (EH2), position 11 to 19 of SEQ ID NO: 2        (EH3), position 20 to 28 of SEQ ID NO: 2 (EH4), position 37 to        45 of SEQ ID NO: 2 (EH5), position 70 to 78 of SEQ ID NO: 2        (EH6), position 73 to 81 of SEQ ID NO: 2 (EH7), position 83 to        91 of SEQ ID NO: 2 (EH8), position 107 to 115 of SEQ ID NO: 2        (EH9), position 110 to 118 of SEQ ID NO: 2 (EH10), and    -   the T cell epitopes of the light chain immunoglobulin variable        domain at position 2 to 10 of SEQ ID NO: 12 (EL1), position 3 to        11 of SEQ ID NO: 12 (EL2), position 10 to 18 of SEQ ID NO: 12        (EL3), position 11 to 19 of SEQ ID NO: 12 (EL4), position 45 to        53 of SEQ ID NO: 12 (EL5), position 53 to 61 of SEQ ID NO: 12        (EL6), position 59 to 67 of SEQ ID NO: 12 (EL7), position 61 to        69 of SEQ ID NO: 12 (EL8), position 62 to 70 of SEQ ID NO: 12        (EL9), position 70 to 78 of SEQ ID NO: 12 (EL10), and position        97 to 105 of SEQ ID NO: 12 (EL11).-   7. The antibody of any one of items 1 to 6, wherein for removing    said at least one T cell epitope at least one amino acid within said    at least one T cell epitope is substituted by another amino acid.-   8. The antibody of item 7, wherein the substitution is selected from    the group consisting of    -   T9S, V10E, A12K, Q19K, S28T, K38R, R40A, L70I, A72R, V73D, S91T,        T115L, L116V in SEQ ID NO: 2, and    -   I10T, M11L, L46A, V59S, I62S, S69D, R76S, L105I in SEQ ID NO:        12.-   9. The antibody of any one of items 6 to 8,    -   wherein 0, 1, 2, or 3 substitutions are within each of EH1 and        EH6;    -   wherein 0, 1 or 2 substitutions are within each of EH2, EH5, and        EH10;    -   wherein 0 or 1 substitutions are within each of EH3, EH4, EH7,        EH8, and EH9;    -   wherein 0, 1 or 2 substitutions are within each of EL2, EL3,        EL7, EL8, and EL9; and    -   wherein 0 or 1 substitutions are within each of EL1, EL4, EL5,        EL6, EL10, and EL11, with the proviso that at least one        substitution is present.-   10. The antibody of any one of items 1 to 9, wherein 6, 8, or 10 T    cell epitopes are removed from the heavy chain immunoglobulin    variable domain, and/or    -   wherein 5, 9, 10, or 11 T cell epitopes are removed from the        light chain immunoglobulin variable domain.-   11. The antibody of any one of items 1 to 10, wherein the heavy    chain immunoglobulin variable domain comprises a sequence selected    from the group consisting of SEQ ID NOs: 4, 6, 8, and 10; and/or    -   wherein the light chain immunoglobulin variable domain comprises        a sequence selected from the group consisting of SEQ ID NOs: 14,        16, 18, and 20.-   12. The antibody of item 10 or 11, wherein    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 4 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 14;    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 4 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 20;    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 6 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 14;    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 6 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 16;    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 6 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 20;    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 8 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 16;    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 8 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 20;    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 10 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 14;    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 10 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 16; or    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 10 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 20.-   13. The antibody of item 12, wherein    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 4 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 14;    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 6 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 16;    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 10 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 16; or    -   the heavy chain immunoglobulin variable domain comprises a        sequence identical to SEQ ID NO: 10 and the light chain        immunoglobulin variable domain comprises a sequence identical to        SEQ ID NO: 20.-   14. The antibody of any one of items 1 to 13, wherein the binding    affinity of the antibody to CD4 is within one order of magnitude    higher or lower than the binding affinity of the antibody produced    by the hybridoma cell line ECACC 88050502.-   15. The antibody of item 14, wherein the binding affinity of the    antibody to CD4 is within twofold higher or lower than the binding    affinity of the antibody produced by the hybridoma cell line ECACC    88050502.-   16. The antibody of item 14 or 15, wherein the antibody has a higher    binding affinity to CD4 than the antibody produced by the hybridoma    cell line ECACC 88050502.-   17. The antibody of any one of items 1 to 16,    -   (a) wherein the antibody has a reduced ability to bind to MHC        class II molecules;    -   (b) wherein the antibody induces a weaker immune response in a        subject;    -   (c) wherein the protein is a full-length antibody;    -   (d) wherein the antibody is a chimeric antibody; and/or    -   (e) wherein the substitution(s) is/are within the most        immunogenic regions of the parent molecule.-   18. The antibody of any one of items 1 to 17, wherein the heavy    chain variable region is linked to a human IgG4 constant region    domain, and wherein the light chain variable region is linked to a    human kappa constant region domain.-   19. The antibody of item 18, wherein the antibody is obtained using    the expression vectors pANTVhG4 and pANTVκ.-   20. A method of preparing an antibody of any one of items 1 to 19    comprising the following steps    -   (i) providing the amino acid sequence of the antibody derivable        from the hybridoma cell line ECACC 88050502 or part thereof;    -   (ii) identifying one or more T cell epitopes within the amino        acid sequence of the antibody or part thereof by any method        including determination of the binding of the peptides to MHC        molecules using in vitro or in silico techniques or biological        assays;    -   (iii) designing new sequence variants with one or more amino        acids within the identified T cell epitopes modified in such a        way to substantially reduce or eliminate binding of the peptides        to MHC molecules measured by in vitro or in silico techniques or        biological assays; and    -   (iv) constructing such sequence variants by recombinant DNA        techniques and testing said sequence variants in order to        identify one or more sequence variants having the properties of        an antibody of any one of items 1 to 20.-   21. A pharmaceutical composition comprising an antibody of any one    of items 1 to 19 and a pharmaceutically acceptable carrier.-   22. Use of an antibody of any one of items 1 to 19 for the    manufacture of a medicament for therapeutically treating a subject.-   23. The use of item 22, wherein the medicament is for treating an    autoimmune condition, particularly an autoimmune condition selected    from multiple sclerosis, rheumatoid arthritis, systemic vasiculitis,    uveitis, inflammatory bowel disease and scleroderma; or wherein the    medicament is for immunosuppressing a patient prior to or subsequent    to transplantation of an organ, particularly a kidney.-   24. The antibody of any one of items 1 to 19 for use in a method of    therapeutic treatment.-   25. The antibody of item 24, wherein the method is for treating an    autoimmune condition, particularly an autoimmune condition selected    from multiple sclerosis, rheumatoid arthritis, systemic vasiculitis,    uveitis, inflammatory bowel disease and scleroderma; or wherein the    method is for immunosuppressing a patient prior to or subsequent to    transplantation of an organ, particularly a kidney.-   26. The use or antibody of any one of items 22 to 25, wherein said    subject is a human.-   27. A nucleic acid encoding a heavy chain and/or a light chain    immunoglobulin variable domain of an antibody of any one of items 1    to 19.-   28. The nucleic acid of item 27, comprising a sequence selected from    the group consisting of SEQ ID NOs 3, 5, 7, 9, 13, 15, 17, and 19.-   29. A vector comprising a nucleic acid of item 27 or 28.-   30. The vector of item 29, wherein the nucleic acid is operably    linked to an expression control sequence.-   31. The antibody of any one of these items, wherein the antibody is    -   i) antibody 16H5.chimIgG4,    -   ii) an antibody obtainable from a cell line CD4.16H5.chimIgG4        deposited with the DSMZ on Dec. 2, 2011.-   32. A host cell comprising a nucleic acid of item 27 or 28 and/or at    least one vector of any one of items 29 to 31.

33. A method of preparing an antibody of any one of items 1 to 19comprising culturing the host cell of item 32 under conditionspermitting expression under the control of suitable expression controlsequence(s), and purifying said antibody from the medium of the cell.

The present invention further relates to alternative embodiments ofembodiments disclosed herein, where the term “ECACC 88050502” isreplaced by “MAX.16H5/30F16H5”.

Likewise, the present invention further relates to embodiments, wherethe term “cell line ECACC 88050502” as used herein, or an equivalentterm, is replaced by the term “cell line MAX.16H5/30F16H5” or anequivalent term.

The present invention further relates to alternative embodiments ofembodiments disclosed herein, where the term “ECACC 88050502” isreplaced by “CD4.16H5.chimIgG4”. Likewise, the present invention furtherrelates to embodiments, where the term “cell line ECACC 88050502” asused herein, or an equivalent term, is replaced by the term “cell lineCD4.16H5.chimIgG4” or an equivalent term.

In the following, the present invention is illustrated by figures andexamples which are not intended to limit the scope of the presentinvention.

EXAMPLES Example 1

Animals

Donor C57Bl/6 CD4k/o mice, C57Bl/6 wild-type mice and recipient Balb/cwild-type mice were bred at the Animal Facility at the University ofLeipzig. The mice strain was maintained under standardized conditions.The C57Bl/6 CD4k/o mice have a stable C57Bl/6 background, in which themurine CD4 molecule is knocked out and express a human CD4. The CD4transgene includes its own promoter ligated to a murine CD4 enhancerelement thus leading to T cell subset-specific expression. CD8+ cellsare not affected in TTG mice. Furthermore, these mice express theHLA-DR3 molecule in addition to the murine MHC II complex. The TTG micehave complete functional murine immune system which is modified withregard to CD4 and HLA-DR. The mice were fed ad libitum. As donors,C57Bl/6 and Balb/c mice were purchased from Charles River (Sulzfeld,Germany; http://jaxmice.jax.org).

All mice were housed, treated or handled in accordance with theguidelines of the University of Leipzig Animal Care Committee and theRegional Board of Animal Care for Leipzig (animal experimentregistration number 28/08).

Statistic Analysis

All data are presented as means±SD. Statistic analysis and graphicpresentation were made using SigmaPlot 10.0/SigmaStat 3.5 software(SYSTAT, Erkrath, Germany).

Irradiation Protocol

For irradiation of mice the X-Ray apparatus (D3225, Orthovoltage, GulmayMedical, Camberley, UK) was adjusted for animal irradiation. Fiveanimals were irradiated in parallel in a plexiglass container (dividedin five spaces per 0.5 cm×64.0 cm), depending on their weight. Theaverage radiation dose was 8.5 Gy.

Preparation of Bone Marrow Cells and Splenocytes

Bone marrow cells (BMCs) were freshly obtained from tibiae and femorafrom C57Bl/6 CD4k/o mice or C57Bl/6 wild-type mice under sterileconditions. Therefore, the musculature and tendons carefully preparedfrom the bone and distal and proximal ends were removed. With a thinneedle (0.4×19 mm), bone marrow cells were rinsed with sterile PBS andcollected in 50 ml tubes. A single cell suspension was achieved bycareful resuspension through a needle. Following, cells were washed oncein PBS (1×) at 300×g for 10 min and resuspended again in PBS (1×) todetermine cell counts using a counting chamber and staining with Tuerkstaining solution. After this, bone marrow cells were washed once againin PBS (1×) at 300×g for 10 min and the cell pellet was resuspended inDulbecco's modified Eagle's minimal essential medium (DMEM; Perbio,Bonn, Germany) without FCS. For the generation of a single cellsplenocyte suspension from C57Bl/6 CD4k/o mice or C57Bl/6 wild-typemice, the spleen were removed immediately under sterile conditions afterdead of mice, pressed through a cell strainer (100 μm) and collected ina 50 ml tube in PBS (1×). The single cell suspension was washed twice inPBS (1×) at 300×g for 10 min. Subsequently, the erythrocytes were lysedin lysis buffer containing 0.155 mol NH₄Cl, 0.01 mol KHCO₃ and 0.01 molEDTA-Na (pH 7.3) in sterile PBS. Cells were washed again, resuspended inDMEM culture medium without FCS and the cell number was determined. Bonemarrow cells and splenocytes were adjusted to the desired cell numberbefore antibody incubation.

As will be readily understood by the skilled person, the use ofsplenocytes in the present Examples occurs for operational reasons—anadditional application of splenocytes is actually not required accordingto the invention, particularly as far as human subjects are concerned.

Antibody Incubation

For the antibody incubation the needed amount of Max 16H5 antibody wasdissolved just before use to a final concentration of 1 mg/ml in DMEM(without FCS). Following, 1.4×10⁸ of bone marrow cells and 1.4×10⁸ ofsplenocytes from C57Bl/6 CD4k/o or C57Bl/6 wild-type mice were incubatedwith 800 μg Max16H5 in 15 ml DMEM without FCS for 1 h at roomtemperature in the dark. As control, bone marrow cells and splenocytesof C57Bl/6 CD4k/o mice or C57Bl/6 wild-type mice without antibodytreatment were also incubated in DMEM without FCS under the sameconditions. After 1 h of incubation cells were centrifuged at 300×g for10 min to pellet them and washed once in PBS (1×) at 300×g for 10 min toremove unbound antibodies.

Cell Transplantation

For co-transplantation experiments 2×10⁷ bone marrow cells of CD4k/omice treated with Max16H5 were added to 2×10⁷ splenocytes of CD4k/o micetreated with Max16H5. The cell concentration was adjusted in a finalvolume of 150 μl sterile 0.9% NaCl. The same was done for bone marrowcells and splenocytes of C57Bl/6 wild-type mice also treated withMax16H5. As a control, 2×10⁷ untreated bone marrow cells of CD4k/o micewere added to 2×10⁷ untreated splenocytes of CD4k/o mice or 2×10⁷ bonemarrow cells of C57Bl/6 wild-type mice were added to 2×10⁷ splenocytesof C57Bl/6 wild-type mice. Following, the grafts were allogeneictransplanted by intravenous injection into the lateral tail vein oflethally irradiated recipient Balb/c wild-type mice. Survival, GvHDsymptoms according Cooke et. al, 1996, and weights were assessed everyday after transplantation.

Flow Cytometry

Before and after transplantation, recipient Balb/c wild-type mice wereanalyzed by flow cytometry.

Characterization of Splenocytes and Bone Marrow Cells of Donor CD4k/oMice.

For cytometric analysis, cells were incubated with 2.5 μl of conjugatedmonoclonal antibodies (murine CD3-FITC, human CD4-APC [both BeckmanCoulter, Krefeld, Germany]; murine CD8-PerCP, MHC-I (H-2D[b])-PE, murineCD4-PECy7, murine CD19-APCCy7 [BD Biosciences, Heidelberg, Germany]). A20 minutes incubation was followed by two washing steps in PBS/1% FBS(1250 rpm, 5 minutes, room temperature [RT]). Finally the pellet wasresuspended with 200 μl of PBS. Additionally, the viability ofsplenocytes and bone marrow cells was tested before transplantation bystaining with 7-Amino-Actinomycin D (7AAD). 1×10⁶ cells were incubatedwith 5 μl (0.25 μg/test) of 7AAD in 300 μl PBS for 30 min at roomtemperature and immediately measured. Data was acquired on a BDFACSCantoII™ Flow Cytometer and analysed using the BD FACSDIVA™ software(both BD Biosciences, Heidelberg, Germany).

Flow Cytometry and Hematology of Recipient Balb/c Wild-Type Mice.

Before and after transplantation procedure, recipient mice were analyzedby flow cytometry. At particular time points, blood (150 μl) was takenfrom the retro orbital vein of each mouse under ether anaesthesia. Bloodwas collected through heparinized capillaries (Greiner Biochemica,Flacht, Germany). Hemoglobin concentration was determined using anAnimal Blood Counter (SCIL, Viernheim, Germany), which had beencalibrated for mouse blood within 2 hours after blood taking. Forcytometric analysis 100 μl of blood cells were incubated with 2.5 μl ofconjugated monoclonal antibodies according to samples (murine CD4-PECy7,MHC-I (H-2D[b])-PE, MHC-I (H-2K[d])-FITC, murine CD8-PerCP, murineCD19-APCCy7 [BD Biosciences, Heidelberg, Germany]; murine CD3-FITC,human CD4-APC [Beckman Coulter, Krefeld, Germany]; human HLA-DR3-FITC[Immunotools, Friesoythe, Germany]. 20 minutes incubation was followedby erythrocyte lysing according to manufacturers instructions (BD FACSLysing Solution [BD Biosciences, Heidelberg, Germany]). By adding ofPBS/1% FBS samples were washed twice (1250 rpm, 5 minutes, roomtemperature [RT]). Finally, the pellet was resuspended with 200 μl ofPBS. For cytometric analysis of murine FoxP3 for detection of regulatoryT cells the murine T_(reg) Detection Kit (Miltenyi Biotec GmbH, BergischGladbach, Germany) was used. 1×10⁶ cells were resuspended in 90 μl MACSBuffer (0.5% FCS, 2 mM EDTA in PBS, [Miltenyi Biotec, Bergisch Gladbach,Germany]) and cell surface markers were stained with 10 μl CD4-FITC andCD25-PE antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany). Afterincubation in the dark for 10 min at 4° C. cells were washed with 2 mlMACS buffer and centrifuged at 300×g for 5 min at 4° C. After removingof the supernatant 1×10⁶ cells were permeabilized by incubation for 30min at 4° C. in a 1 ml of a freshly prepared fixation/permeabilizationsolution (containing formaldehyde). Cells were washed in 2 ml cold MACSbuffer by centrifugation 300×g for 5 min at 4° C. For intracellularFoxP3 staining a permabilzation step was followed after removing of thesupernatant. 1×10⁶ cells were washed with 2 ml of a coldpermeabilization buffer and centrifuged at 300×g for 5 min at 4° C. Thecell pellet was resuspended in 80 μl of cold permeabilization buffer andincubated for 5 min at 4° C. Following, 10 μl of anti-FoxP3-APC(Miltenyi Biotec, Bergisch Gladbach, Germany) antibody was added,carefully mixed and incubated for 30 min at 4° C. Cells were washed with2 ml of a cold permeabilization buffer and centrifuged at 300×g for 5min at 4° C., supernatant was removed and cells were resuspended in 100μl MACS buffer. Data were acquired on a BD FACSCantoII™ Flow Cytometerand analysed using the BD FACSDIVA™ software (both BD Biosciences,Heidelberg, Germany).

Immunohistology

Organs of mice were put in a stainless steel beaker (containing2-methylbutane; Carl Roth, Karlsruhe, Germany), submerged in liquidnitrogen for 15 min and stored at −80° C. until ready for sectioning.Sectioning was done using a Cryostat (Leica Biosystems, Nussloch,Germany); objects were transferred onto a superfrost slide (ThermoScientific, Braunschweig, Germany) and stored immediately at −80° C.until immunohistological analysis. The object slides were incubated with0.3% w/v H₂O₂, dissolved in PBS for 10 min in a wet chamber, and thenwashed three times with PBS. Organs were treated with 10% w/v FBS in PBSfor 60 min at RT, shortly washed with PBS, incubated with avidinsolution (Dako North America, Carpinteria, USA) for 10 min and washedwith PBS. The preparations were incubated with biotin solution (DakoNorth America) for 10 min, washed with PBS, and incubated with theprimary antibody anti human CD4 antibody (United States Biological,Mass., USA) or isotype control (Rat IgG1, κ, BD Biosciences, San Diego,USA), diluted 1:100, for 1 h at RT. Next, slides were covered with asecondary antibody (Biotin-conjugated Goat Anti-Rat IgG₁, BDBiosciences, San Diego, USA), diluted 1:100, for 30 min at RT and washedwith PBS. The object slides were covered with Streptavidin-HorseradishPeroxidase (BD Biosciences, San Diego, USA) for 30 min, washed with PBSfor three times (2 min for each washing step) and incubated with DABdilution (BD Biosciences, San Diego, USA) for 5 min until an obviousintensity of color was achieved and then washed three times with ddH2O.The samples were covered with Mayer's hemalaun solution (Merck,Darmstadt, Germany) for 1 min and then washed with tap water for 10 minto visualize blue staining. The object slides passed through anascending alcohol series (40-100% w/v), were incubated with xylene (CarlRoth) for 5 min and finally covered with Entellan® (Merck). Slides wereanalyzed under microscope (Zeiss, Axio, Imager A1, objective lenses 920EX Plan-Neofluar, Axiocam MRc5 Zeiss, AxioVision Release 4.6.3;Gottingen, Germany).

Histology

Liver, bones and gut of all transgenic mice were analyzedhistologically. Organs were prepared immediately after death andtransferred into formalin (4% w/v; Merck) for hematoxylin-eosin (HE) andkaoline-aniline-orange G (KAO) staining. The formalin boxes were kept inthe dark to prevent formalin precipitation. Bones were incubated inOsteosoft® for at least 7 days at room temperature. All samples wereflushed with tap water for 2 h and then submerged in alcohol dilutionsfrom 70 to 100% w/v for 9 h. The final incubation was with isopropanol(JT Baker, Deventer, The Netherlands) for 1 h and overnight withmethylbenzoate (Riedel de-Häen, Seelze, Germany). After that, the organswere embedded in paraffin for 3 days and sliced (6 lm). The slides wereincubated twice with xylene for 5 min at RT, passed through a descendingalcohol series (100-50% w/v), and finally transferred into ddH2O at RT.Object slides were placed in Mayer's hemalaun solution for 5 min andwashed with tap water for 10 min to reach a blue staining. Afterincubation with 1% w/v eosin Y, the slides passed through an ascending(70-100% w/v) alcohol series and were finally covered with Entellan®(Merck). The object slides were analyzed under the microscope (Nikon,Eclipse TE2000-E 920, objective lenses Plan Fluor 920/0.45 Ph1 DM ∞/0-2WD 7.4 Histo, Software Nikon, LuciaG 5.00; Düsseldorf, Germany). Boneswere stained with KAO as described according to Halmi-Konecny.

Example 2

Recombinant DNA techniques were performed using methods well known inthe art and, as appropriate, supplier instructions for use of enzymesused in these methods. Sources of general methods included standardliterature such as well-known books edited by Sambrook and Russel and byAusubel. Detailed laboratory methods are also described below. In silicomethods such as those described in WO9859244 were used to analyze thevariable heavy and light chain sequences of mouse anti-CD4 for peptidespredicted to bind to MHC Class II molecules (these were considered as Tcell epitopes).

Example 2a: Chimeric Anti-CD4 Antibody

mRNA was extracted from the mouse anti-CD4 hybridoma cells using a PolyA Tract System 1000 mRNA extraction kit: (Promega Corp. Madison Wis.)according to manufacturer's instructions. mRNA was reverse transcribedas follows: For the kappa light chain, 5.0 microliter of mRNA was mixedwith 1.0 microliter of 20 pmol/microliter MuIgκVL-3′ primer OL040 (Table2) and 5.5 microliter nuclease free water (Promega Corp. Madison Wis.).For the lambda light chain, 5.0 microliter of mRNA was mixed with 1.0microliter of 20 pmol/microliter MuIgκVL-3′ primer OL042 (Table 2) and5.5 microliter nuclease free water (Promega Corp. Madison Wis.). For thegamma heavy chain, 5 microliter of mRNA was mixed with 1.0 microliter of20 pmol/microliter MuIgVH-3′ primer OL023 (Table 1) and 5.5 microliternuclease free water (Promega Corp. Madison Wis.). All three reactionmixes were placed in the pre-heated block of the thermal cycler set at70° C. for 5 minutes. These were chilled on ice for 5 minutes beforeadding to each 4.0 microliter ImPromII 5× reaction buffer (Promega Corp.Madison Wis.), 0.5 microliter RNasin ribonuclease inhibitor (PromegaCorp. Madison Wis.), 2.0 microliter 25 mM MgCl2 (Promega Corp. MadisonWis.), 1.0 microliter 10 mM dNTP mix (Invitrogen, Paisley UK) and 1.0microliter Improm II reverse transcriptase (Promega Corp. Madison Wis.).The reaction mixes were incubated at room temperature for 5 minutesbefore being transferred to a pre-heated PCR block set at 42° C. for 1hour. After this time the reverse transcriptase was heat inactivated byincubating at 70° C. in a PCR block for fifteen minutes.

Heavy and light chain sequences were amplified from cDNA as follows: APCR master mix was prepared by adding 37.5 microliter 10× Hi-Fi ExpandPCR buffer: (Roche, Mannheim Germany), 7.5 microliter 10 mM dNTP mix(Invitrogen, Paisley UK) and 3.75 microliter Hi-Fi Expand DNA polymerase(Roche, Mannheim Germany) to 273.75 microliter nuclease free water. Thismaster mix was dispensed in 21.5 microliter aliquots into 15 thin walledPCR reaction tubes on ice. Into six of these tubes was added 2.5microliter of MuIgVH-3′ reverse transcription reaction mix and 1.0microliter of heavy chain 5′ primer pools HA to HF (see Table 1 forprimer sequences and primer pool constituents). To another seven tubeswas added 2.5 microliter of MuIgκVL-3′ reverse transcription reactionand 1.0 microliter of light chain 5′ primer pools LA to LG (Table 2).Into the final tube was added 2.5 microliter of MuIgκVL-3′ reversetranscription reaction and 1.0 microliter of lambda light chain primerMuIgλVL5′-LI. Reactions were placed in the block of the thermal cyclerand heated to 95° C. for 2 minutes. The PCR reaction was performed for40 cycles of 94° C. for 30 seconds, 55° C. for 1 minute and 72° C. for30 seconds. Finally the PCR products were heated at 72° C. for 5minutes, and then held at 4° C.

TABLE 1 Code Sequence Length Name-Pool OL007 ATGRASTTSKGGYTMARCTKGRTTT25 MuIgV_(H)5′-HA OL008 ATGRAATGSASCTGGGTYWTYCTCTT 26 MuIgV_(H)5′-HBOL009 ATGGACTCCAGGCTCAATTTAGTTTTCCT 29 MuIgV_(H)5′-HC OL010ATGGCTGTCYTRGBGCTGYTCYTCTG 26 MuIgV_(H)5′-HC OL011ATGGVTTGGSTGTGGAMCTTGCYATTCCT 29 MuIgV_(H)5′-HC OL012ATGAAATGCAGCTGGRTYATSTTCTT 26 MuIgV_(H)5′-HD OL013ATGGRCAGRCTTACWTYYTCATTCCT 26 MuIgV_(H)5′H-D OL014ATGATGGTGTTAAGTCTTCTGTACCT 26 MuIgV_(H)5′-HD OL015ATGGGATGGAGCTRTATCATSYTCTT 26 MuIgV_(H)5′-HE OL016ATGAAGWTGTGGBTRAACTGGRT 23 MuIgV_(H)5′-HE OL017ATGGRATGGASCKKIRTCTTTMTCT 25 MuIgV_(H)5′-HE OL018ATGAACTTYGGGYTSAGMTTGRTTT 25 MuIgV_(H)5′-HF OL019ATGTACTTGGGACTGAGCTGTGTAT 25 MuIgV_(H)5′-HF OL020ATGAGAGTGCTGATTCTTTTGTG 23 MuIgV_(H)5′-HF OL021ATGGATTTTGGGCTGATTTTTTTTATTG 28 MuIgV_(H)5′-HF OL023CCAGGGRCCARKGGATARACIGRTGG 26 MuIgV_(H)3′-2 (SEQ ID NOs: 70-85)

TABLE 2 Code Sequence Length Name-Pool OL024 ATGRAGWCACAKWCYCAGGTCTTT 24MuIgkV_(L)5′-LA OL025 ATGGAGACAGACACACTCCTGCTAT 25 MuIgkV_(L)5′-LB OL026ATGGAGWCAGACACACTSCTGYTATGGGT 29 MuIgkV_(L)5′-LC OL027ATGAGGRCCCCTGCTCAGWTTYTTGGIWTCTT 32 MuIgkV_(L)5′-LD OL028ATGGGCWTCAAGATGRAGTCACAKWYYCWGG 31 MuIgkV_(L)5′-LD OL029ATGAGTGTGCYCACTCAGGTCCTGGSGTT 29 MuIgkV_(L)5′-LE OL030ATGTGGGGAYCGKTTTYAMMCTTTTCAATTG 31 MuIgkV_(L)5′-LE OL031ATGGAAGCCCCAGCTCAGCTTCTCTTCC 28 MuIgkV_(L)5′-LE OL032ATGAGIMMKTCIMTTCAITTCYTGGG 26 MuIgkV_(L)5′-LF OL033ATGAKGTHCYCIGCTCAGYTYCTIRG 26 MuIgkV_(L)5′-LF OL034ATGGTRTCCWCASCTCAGTTCCTTG 25 MuIgkV_(L)5′-LF OL035ATGTATATATGTTTGTTGTCTATTTCT 27 MuIgkV_(L)5′-LF OL036ATGAAGTTGCCTGTTAGGCTGTTGGTGCT 29 MuIgkV_(L)5′-LG OL037ATGGATTTWCARGTGCAGATTWTCAGCTT 29 MuIgkV_(L)5′-LG OL038ATGGTYCTYATVTCCTTGCTGTTCTGG 27 MuIgkV_(L)5′-LG OL039ATGGTYCTYATVTTRCTGCTGCTATGG 27 MuIgkV_(L)5′-LG OL040ACTGGATGGTGGGAAGATGGA 21 MuIgkV_(L)3′-1 OL041 ATGGCCTGGAYTYCWCTYWTMYTCT25 MuIgλV_(L)5′-LI OL042 AGCTCYTCWGWGGAIGGYGGRAA 23 MuIgλV_(L)3′-1 (SEQID NOs: 86-104)

Amplification products were cloned into pGEM-T easy vector using thepGEM-T easy Vector System I (Promega Corp. Madison Wis.) kit andsequenced. The resultant mouse VH and VL sequences are shown as SEQ IDNOs: 1 and 2 (FIG. 12A) and SEQ ID NOs: 11 and 12 (FIG. 13A).

For generation of a chimeric antibody, VH region genes were amplified byPCR using the primers OL330 and OL331 (Table 3); these were designed toengineer in a 5′ MluI and a 3′ HindIII restriction enzyme site usingplasmid DNA from one of the cDNA clones as template. Into a 0.5 ml PCRtube was added 5 microliter 10×Hi-Fi Expand PCR buffer: (Roche, MannheimGermany), 1.0 microliter 10 mM dNTP mix (Invitrogen, Paisley UK), 0.5microliter of Primer OL330, 0.5 microliter of primer OL331, 1.0microliter template DNA and 0.5 microliter Hi-Fi Expand DNA polymerase(Roche, Mannheim Germany) to 41.5 microliter nuclease free water.

TABLE 3 Code Sequence Length OL 330GATCACGCGTGTCCACTCCGAAGTGCAGCTGGTGGAGTC 39 OL 331GTACAAGCTTACCTGAGGAGACGGTGACTGAGG 33 (SEQ ID NOs: 105-106)

VL regions were amplified in a similar method using the oligonucleotidesOL332 and OL333 (Table 4) to engineer in BssHII and BamHI restrictionenzyme sites. Reactions were placed in the block of the thermal cyclerand heated to 95° C. for 2 minutes. The polymerase chain reaction (PCR)reaction was performed for 30 cycles of 94° C. for 30 seconds, 55° C.for 1 minute and 72° C. for 30 seconds. Finally the PCR products wereheated at 72° C. for 5 minutes, and then held at 4° C. VH and VL regionPCR products were then cloned into the vectors pANTVhG4 and pANTVκrespectively (FIG. 11) at the MluI/HinDIII and BssHII/BamHI sitesrespectively. Both pANTVhG4 and pANTVκ are pAT153-based plasmidscontaining a human Ig expression cassette. The heavy chain cassette inpANTVhG4 consists of a human genomic IgG4 constant region gene driven byhCMVie promoter, with a downstream human IgG polyA region. pANTVhG4 alsocontains a hamster dhfr gene driven by the SV40 promoter with adownstream SV40 polyA region.

The light chain cassette of pANTVκ is comprised of the genomic humankappa constant region driven by hCMVie promoter with downstream lightchain polyA region. Cloning sites between a human Ig leader sequence andthe constant regions allow the insertion of the variable region genes.

TABLE 4 Code Sequence Length OL 332 CATGGCGCGCGATGTGACATCCAGATGACTCAGTC35 TGCGGGATCCAACTGAGGAAGCAAAGTTTAAATTCTACTCACGT OL 333CTCAGCTCCAGCTTGGTCC 63 (SEQ ID NOs: 107-108)

NS0 cells (ECACC 85110503, Porton, UK) were co-transfected with thesetwo plasmids via electroporation and selected in DMEM (Invitrogen,Paisley UK)+5% FBS (Ultra low IgG Cat No. 16250-078 Invitrogen, PaisleyUK)+Penicillin/Streptomycin (Invitrogen, Paisley UK)+100 nM Methotrexate(Sigma, Poole UK). Methotrexate resistant colonies were isolated andantibody was purified by Protein A affinity chromatography using a 1 mlHiTrap MabSelect Sure column (GE Healthcare, Amersham UK) following themanufacturers recommended conditions.

NS0 supernatants were quantified for antibody expression in IgG Fc/KappaELISA using purified human IgG1/Kappa (Sigma, Poole UK) as standards.Immunosorb 96 well plates (Nalgene Hereford, UK) were coated with mouseanti-human IgG Fc-specific antibody (I6260 Sigma, Poole UK) diluted at1:1500 in 1×PBS (pH 7.4) at 37° C. for 1 hour. Plates were washed threetimes in PBS+0.05% Tween 20 before adding samples and standards, dilutedin 2% BSA/PBS. Plates were incubated at RT for 1 hour before washingthree times in PBS/Tween and adding 100 μl/well of detecting antibodygoat anti-human kappa light chain peroxidase conjugate (A7164 Sigma,Poole UK) diluted 1:1000 in 2% BSA/PBS. Plates were incubated at RT for1 hour before washing five times with PBS/tween and bound antibodydetected using OPD substrate (Sigma, Poole UK). The assay was developedin the dark for 5 minutes before being stopped by the addition of 3MHCl. The assay plate was then read in a MRX TCII plate reader (DynexTechnologies, Worthing, UK) at 490 nm.

The chimeric antibody was tested in an ELISA-based competition assayusing mouse anti-CD4 antibody, biotinylated using a B-Tag microbiotinylation kit (Sigma, Poole UK). A dilution series of chimeric IgG4or control mouse antibody from 10 μg/ml to 0.009 μg/ml was premixed witha constant concentration of biotinylated anti-CD4 (0.2 μg/ml) beforeincubating 100 μl/well for 1 hour at room temperature in a Nunc MaxiSorp96 well flat bottom microtitre plate (Fisher, Loughborough, UK)pre-coated with 50 μl/well of 1 μg/ml CD4. The binding of thebiotinylated mAb was determined by incubating for 1 h at roomtemperature with 100 μl/well of a 1/500 dilution of streptavidin-HRP(Sigma), followed by detection with 100 μl/well OPD substrate (Sigma).After stopping the reaction with 50 μl/well 3M HCl, absorbance at 490 nmwas measured using a Dynex Technologies (Worthing, UK) MRX TC II platereader.

The results obtained (FIG. 14) show that the chimeric IgG4 and mouseanti-CD4 antibodies have very similar binding profiles, with IC50 valuesof 0.25 μg/ml and 0.18 μg/ml respectively. Therefore the correctvariable region sequences have been identified and cloned.

Example 2b: Design of Modified Anti-CD4 Antibodies

Sequential 9mer peptides spanning the entire length of the variableregions were tested in silico for binding against a panel of 34 MHCclass II alleles. The scores for each individual allele were normalizedto a scale of 0 to 1 and extensive validation with panels of known MHCclass II binding peptides has demonstrated that a cut-off value of 0.55effectively discriminates between predicted binding and non-bindingpeptides. In detail, potential MHC class II binding sequences within thevariable domains were identified using the software iTope™. The iTope™software predicts favorable interactions between amino acid side chainsof a peptide and specific binding pockets within the binding grooves of34 human MHC class II alleles. The location of key binding residues isachieved by the in silico generation of 9mer peptides that overlap byone amino acid spanning the test protein sequence. Each 9mer was scoredbased on the potential ‘fit’ and interactions of amino acid side chainswith the binding groove of the MHC class II molecules. The peptidescores calculated by the software lie between 0 and 1. Peptides thatproduced a high mean binding score (>0.55 in the iTope™ scoringfunction) were highlighted and, if >50% of the MHC class II bindingpeptides, i.e. 17 out of 34 alleles had a high binding affinity(score >0.6), such peptides were defined as “promiscuous high affinity”MHC class II binding peptides which are considered a high risk forcontaining CD4⁺ T cell epitopes. Moderate affinity MHC class II bindingpeptides bind a high number of alleles with high affinity but less than17. High and moderate affinity binders were then analyzed using theiTope™ software for changes that could be made to the sequence thatwould reduce or remove binding to the MHC class II alleles. In makingamino-acid selections, the range of amino-acids naturally found in humanantibodies at any given position was considered. Alternatively, publiclyavailable softwares could be used, for example:

ProPred (www.imtech.res.in/raghava/propred/),

Rankpep (bio.dfci.harvard.edu/RANKPEP/) or

NetMHCII (www.cbs.dtu.dk/services/NetMHCII/)

Ten MHC class II binding peptides were identified in the mouse heavychain variable region (Table 5) and eleven identified in the mouse lightchain variable region (Table 6). Tables 5 and 6 list the identified MHCclass II binding sequences and the series of sequence variants that wereused in the sequences of FIGS. 12A, 12B, 12C, 12D, and 12E and FIGS.13A, 13B, 13C, 13D, and 13E in order to reduce MHC class II binding.Where MHC class II binding sequences were identified, amino acids in thepeptide at key MHC class II binding positions were replaced withalternative amino acids in order to reduce or eliminate MHC class IIbinding. In some instances, more than one mutation was required toremove binding completely and, in other instance, MHC class II bindingcould not be completely removed although the numbers of alleles involvedwere small and the binding scores were close to the cut-off value.

TABLE 5 Mouse VH4 VH3 VH2 VH1 Sequence * Sequence * Sequence *Sequence * Sequence * LQQSGTVLA 26 LQQSGTELK 13 LQQSGSELK  0 LQQSGSELK 0LQQSGSELK 0 VLARPGASV 29 ELKRPGASV  0 ELKRPGASV  0 ELKRPGASV 0 ELKRPGASV0 LARPGASVQ 20 LKRPGASVK  2 LKRPGASVK  2 LKRPGASVK 2 LKRPGASVK 2MSCKASGYS 18 MSCKASGYT  7 MSCKASGYT  7 VSCKASGYT 0 VSCKASGYT 0 VKQRPGQGL24 VKQAPGQGL  1 VKQAPGQGL  1 VKQAPGQGL 1 VRQAPGQGL 1 LTAVTSAST 17LTAVTSAST 17 LTAVTSAST 17 LTADTSAST 4 ITRDTSAST 0 VTSASTAYM 31 VTSASTAYM31 VTSASTAYM 31 DTSASTAYM 0 DTSASTAYM 0 LSSLTNEDS 20 LSSLTNEDS 20LSSLTNEDT  2 LSSLTNEDT 2 LSSLTNEDT 2 LDYWGQGTT 21 LDYWGQGTT 21 LDYWGQGTL 0 LDYWGQGTL 0 LDYWGQGTL 0 WGQGTTLTV 27 WGQGTTVTV  0 WGQGTLVTV  0WGQGTLVTV 0 WGQGTLVTV 0 SEQ ID SEQ ID NOs: 42-55, 138 NOs: 21-30 (*)Number of alleles bound

TABLE 6 Mouse VK4 VK3 VK2 VK1 Sequence * Sequence * Sequence *Sequence * Sequence * IVLTQSPAI 27 IVLTQSPAI 27 IVLTQSPAT  3 IVLTQSPAT 3IVLTQSPAT 3 VLTQSPAIM 11 VLTQSPAIM 11 VLTQSPATL  0 VLTQSPATL 0 VLTQSPATL0 IMSASPGEK 10 IMSASPGEK 10 TLSASPGEK  0 TLSASPGEK 0 TLSASPGEK 0MSASPGEKV 24 MSASPGEKV 24 LSASPGEKV  2 LSASPGEKV 2 LSASPGEKV 2 LLIYDTSNL 6 LLIYDTSNL  6 LLIYDTSNL  6 LLIYDTSNL 6 ALIYDTSNL 0 LASGVPVRF 19LASGVPSRF  2 LASGVPSRF  2 LASGVPSRF 2 LASGVPSRF 2 VRFIGSGSG 29 SRFIGSGSG 0 SRFIGSGSG  0 SRFSGSGSG 0 SRFSGSGSG 0 FIGSGSGTS 14 FIGSGSGTD  0FIGSGSGTD  0 FSGSGSGTD 0 FSGSGSGTD 0 IGSGSGTSY 22 IGSGSGTDY 22 IGSGSGTDY22 SGSGSGTDY 0 SGSGSGTDY 0 YSLTISRME 17 YSLTISSME  0 YSLTISSME  0YSLTISSME 0 YSLTISSME 0 FGAGTKLEL 16 FGAGTKLEI  0 FGAGTKLEI  0 FGAGTKLEI0 FGAGTKLEI 0 SEQ ID SEQ ID NOs: 56-69 NOs: 31-41 (*) Number of allelesbound

Example 2c: Generation of Modified Anti-CD4 Antibodies

Initial modified antibody VH and VK region genes were generated byoverlap PCR mutagenesis of the parental mouse anti-CD4 variable regionsfrom example 2a (SEQ ID NO: 1 (FIG. 12A) and SEQ ID NO: 11 (FIG. 13A)).(FIGS. 12A, 12B, 12C, 12D, and 12E and FIGS. 13A, 13B, 13C, 13D, and13E) using methods known in the art. Further variants were constructedby overlap PCR mutagenesis from SEQ ID NO: 3 (FIG. 12B) and SEQ ID NO:13 (FIG. 13B). The assembled variants were then cloned directly into theexpression vectors of FIG. 11. All clones were verified by DNAsequencing. In detail, PCR mutagenesis was performed as follows: Primerpairs were designed that spanned the region of the template nucleotidesequence that was to be altered, on both sense and anti-sense DNAstrands. The primers contained the sequence that was to beintroduced/altered, flanked by sequences that were identical to thetemplate sequence and served to anchor the primers in the correctlocation. 5′ and 3′ end primers were also required that containedrestriction sites suitable for cloning the mutated PCR product into theexpression vectors (i.e. MluI and HinDIII for the VH gene and BssHII andBamHI for the VK gene). Tables 3, 4 and 7 list all the primers used forthe construction of the de-immunized variants. For overlap PCR, smallfragments of the gene are PCR amplified individually that overlapneighboring fragments at their ends, and mutations are introduced by theprimers at the regions of overlap. The short PCR fragments are thenpurified and assembled together in a single PCR reaction using 5′ and 3′end primers. To create the variant 4 genes, the murine variable regionswere used as template and for all subsequent variants, the variant 4genes were used as template for the PCR reactions: For VH variant 4, twoindividual PCRs were performed using OL335+OL337 and OL336+OL338, andthe fragments joined using OL334+OL338. For VH variant 3, two individualPCRs were performed using OL339+OL341 and OL340+OL342, and the fragmentsjoined using OL339+OL342. For VH variant 2, three individual PCRs wereperformed using OL330+OL344, OL343+OL346 and OL354+OL342, and thefragments joined using OL330+OL342. For VH variant 1, three individualPCRs were performed using OL339+OL348, OL347+OL350 and OL349+OL342, andthe fragments joined using OL339+OL342. For VK variant 4, fourindividual PCRs were performed using OL332+OL352, OL351+OL354,OL353+OL356, and OL355+OL357 and the fragments joined using OL332+OL357.For VK variant 3, one PCR was performed using OL358+OL357. For VKvariant 2, two individual PCRs were performed using OL358+OL360 andOL359+OL357, and the fragments joined using OL358+OL357. For VK variant1, two individual PCRs were performed using OL358+OL362 and OL361+OL357,and the fragments joined using OL358+OL357. The PCR conditions used wereas described in Example 2a for the amplification of the chimericvariable region genes. The PCR fragments generated were digested witheither MluI and HinDIII for the VH genes or BssHII and BamHI for theVKgenes and cloned into the appropriate expression vectors.

TABLE 7 Code Sequence Length OL334GTTGCTACGCGTGTCCACTCCGAGGTTCAGCTCCAGCAGTCTGGGACTGaGCTGaaAAGGCCTGG 65OL335 ACTGaGCTGaaAAGGCCTGGGGCTTCCGTGaAGATGTCCTGCAAGGCTTCTGGCTACAcCTTTGC65 OL336 ACAGGGTCTACAATGGATTGG 21 OL337CCAATCCATTGTAGACCCTGTCCAGGggcCTGTTTTA 37 OL338CCCAGAAAGCTTACCTGAGGAGACTGTGAcAGTGGTGCC 39 OL339GTTGCTACGCGTGTCCACTCCGAGGTTCAGCTCCAGCAGTCTGGGtCTGAGCTGAAAAGG 60 OL340CAAATGAGGACaCcGCGGTCTATT 24 OL341 AATAGACCGCgGtGTCCTCATTTG 24 OL342CCCAGAAAGCTTACCTGAGGAGACTGTGACAagGGTGCC 39 OL343CTTCCGTGAAGgTGTCCTGCAAGGC 25 OL344 GCCTTGCAGGACAcCTTCACGGAAG 25 OL345AACTGACTGCAGaCACATCCGCCAG 25 OL346 CTGGCGGATGTGtCTGCAGTCAGTT 25 OL347TGCACTGGGTAAgACAGGCCCCTGG 25 OL348 CCAGGGGCCTGTcTTACCCAGTGCA 25 OL349GTTCAAGGACAAGGCCAAAaTcACTagAGACACATCCGCCAGCACT 46 OL350AGTGCTGGCGGATGTGTCTctAGTgAtTTTGGCCTTGTCCTTGAAC 46 OL351CTCCAGGGGAGAAGGcCGCCATGACC 26 OL352 GGTCATGGCGgCCTTCTCCCCTGGAG 26 OL353TCCTGATTTATGACACATCCAACCTGGCTTCTGGAGTCCCTtcTCGCTTCA 51 OL354GTTGGATGTGTCATAAATCAGGA 23 OL355TCTGGGACCgaTTACTCTCTCACAATCAGCaGcATGGAGGCTG 43 OL356CAGCCTCCATgCtGCTGATTGTGAGAGAGTAAtcGGTCCCAGA 43 OL357ATTGCGGGATCCAACTGAGGAAGCAAAGTTTAAATTCTACTCACGTTTgAtCTCCAGCTTG 61 OL358CCCAGGCGCGCGATGTCAAATTGTTCTCACCCAGTCTCCAGCAAcCCTGTCTGCA 55 OL359TTCTCGCTTCAgcGGCAGTGGG 22 OL360 CCCACTGCCgcTGAAGCGAGAA 22 OL361CTCCCCCAGAgcCCTGATTTAT 22 OL362 ATAAATCAGGgcTCTGGGGGAG 22 (SEQ ID NOs:109-137)

All combinations of variant heavy and light chains (i.e. a total of 16pairings) were stably transfected into NS0 cells via electroporation.Transfected cells were initially selected using 100 nM methotrexate,expanded into 200 nm methotrexate and tested for IgG expression as inexample 2a. No expression was observed with variants possessing the VK3chain or with variants VH3/VK1 and VH1/VK2. The best expressing linesfor each variant were expanded and frozen under liquid nitrogen. Antihuman CD4-antibody variants from the NS0 stable transfections werepurified from cell culture supernatants via protein A affinitychromatography. Supernatants were pH adjusted with 0.1 volumes of 10×PBSpH 7.4 and passed over 1 ml Mab Select Sure Protein A columns (GEHealthcare, Amersham, UK). The columns were washed with 10 volumes ofPBS pH 7.4 before elution with 50 mM citrate buffer pH 3.0. 1 mlfractions were collected and immediately neutralized with 0.1 ml of 1MTris-HCl pH 9.0. Protein containing fractions (as measured by absorbanceat 280 nm) were pooled, buffer exchanged into PBS pH 7.4 and thepurified antibodies stored at +4° C. The concentrations of the purifiedantibodies were measured by UV absorbance at 280 nm. The purifiedantibodies were tested for binding to their target, human CD4 viacompetition ELISA. Nunc MaxiSorp 96 well flat bottom microtitre plates(Fisher) were coated overnight at 4° C. with 50 μl/well of 1 μg/ml CD4in PBS pH 7.4. Duplicate titrations of mouse antibody and epitopedepleted antibody samples were generated (in the range 0.005 μg/ml to 12μg/ml) and mixed with a constant concentration (0.2 μg/ml) ofbiotinylated mouse antibody in PBS pH 7.4/2% BSA. The titrations (finalvolume 100 μl/well) were added to pre-washed (4× with PBS pH 7.4/0.05%Tween 20) assay plates and incubated at room temperature for 1 hour.Plates were then washed as above and 100 μl/well of a 1/500 dilution ofstreptavidin HRP (Sigma) in PBS pH 7.4/0.05% Tween 20 was added andincubated for a further 1 hour at room temperature. After furtherwashing, bound biotinylated mouse antibody was detected with 100 μl/well3,3′-5,5′ tetramethylbenzidine substrate (Sigma). After stopping thereaction with 50 μl/well 3M HCl, absorbance was measured at 450 nm on aDynex Technologies MRX TC II plate reader and the binding curves of thetest antibodies were compared to the mouse reference standard and thepurified chimeric antibody. The results are shown in FIGS. 15A, 15B, and16. Absorbance was plotted against sample concentration and straightlines were fitted through each of the data sets. The equations of thelines were used to calculate the concentration required to inhibitbiotinylated mouse antibody binding to CD4 by 50% (IC50). The IC50values of the test samples were divided by that of the mouse antibody tocalculate the fold difference in binding efficiencies. These values arereported in Table 8, which shows that seven of the antibodies(highlighted in underlining) bind at least within two-fold of the mousereference antibody and four antibodies (VH1/VK1, VH2/VK2, VH4/VK2 andVH4/VK4) show binding that is improved.

TABLE 8 Relative Binding of Anti-CD4 Variants VK1 VK2 VK3 VK4 VH1 0.14 —— 5.06 VH2 8.66 0.96 — 1.44 VH3 — 1.77 — 1.44 VH4 3.89 0.73 — 0.49

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1-16. (canceled)
 17. An anti CD4 antibody, selected from the groupconsisting of: i) antibody 16H5.chimIgG4; ii) an antibody obtainablefrom cell line CD4.16H5.chimIgG4 deposited with the DSMZ with depositnumber DSM ACC3147 on Dec. 2, 2011; iii) an antibody comprising the VHand the VK of antibody 16H5.chimIgG4; iv) an antibody comprising a VHand a VK of an antibody obtainable from a cell line CD4.16H5.chimIgG4deposited with the DSMZ on Dec. 2, 2011; v) an antibody comprising acombination of a VH disclosed in FIG. 12 and of a VK disclosed in FIG.13, wherein said combination is selected from VH1NK1, VH2/VK2, VH4/VK2and VH4/VK4, especially wherein said combination is VH2/VK2.